From the Wellcome Trust Laboratories for Molecular Parasitology, Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom
Received for publication, November 7, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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Co-translational modification of eukaryotic
proteins by N-myristoylation aids subcellular targeting and
protein-protein interactions. The enzyme that catalyzes this process,
N-myristoyltransferase (NMT), has been characterized in the
kinetoplastid protozoan parasites, Leishmania and
Trypanosoma brucei. In Leishmania major, the
single copy NMT gene is constitutively expressed in all
parasite stages as a 48.5-kDa protein that localizes to both membrane
and cytoplasmic fractions. Leishmania NMT myristoylates the
target acylated Leishmania protein, HASPA, when both are
co-expressed in Escherichia coli. Gene targeting
experiments have shown that NMT activity is essential for viability in
Leishmania. In addition, overexpression of NMT causes gross
changes in parasite morphology, including the subcellular accumulation
of lipids, leading to cell death. This phenotype is more extreme than
that observed in Saccharomyces cerevisiae, in which
overexpression of NMT activity has no obvious effects on growth
kinetics or cell morphology. RNA interference assays in T. brucei have confirmed that NMT is also an essential protein in
both life cycle stages of this second kinetoplastid species, suggesting
that this enzyme may be an appropriate target for the development of
anti-parasitic agents.
Myristoyl-CoA:protein N-myristoyltransferase
(NMT1; EC 2.3.1.97) catalyzes
the covalent co-translational attachment of the fatty acid, myristate
(C14:0) to the amino-terminal glycine residue of a number of eukaryotic
cellular and viral proteins of diverse function (1). These include
cAMP-dependent serine/threonine kinases, members of the p60
Src family of tyrosine kinases, retroviral gag polyprotein precursors
such as HIV-1pr55, viral capsid components, and the The NMT enzyme has an ordered Bi-Bi reaction mechanism (5), binding
first to myristoyl-CoA, with the resulting conformational changes
generating a peptide-binding site. Subsequent formation of a ternary
myristoyl-CoA:NMT-peptide complex leads to catalysis and product
release. The myristoyl-CoA binding sites of purified human NMT and the
fungal NMTs are highly conserved, but their peptide binding
specificities are divergent (6). By exploiting these differences,
peptide and peptidomimetic inhibitors with selectivity for fungal NMTs
have been designed. For example, an inhibitor based on the N terminus
of ADP-ribosylation factor (ARF), the major myristoylated protein
detected in [3H]myristate-labeled C. neoformans (causative agent of chronic meningitis in AIDS
patients), is competitive for peptide and noncompetitive for
myristoyl-CoA binding (7), and a nonpeptide inhibitor based on this
sequence is at least ~5-fold selective for C. neoformans against human NMT (8). Similarly, peptidomimetic inhibitors based on
the N-terminal sequence of C. albicans ARF are 560-fold selective for the fungal NMT as compared with the human enzyme. Gel
mobility assays indicate that a single dose of 200 µM is
sufficient to produce ~50% reduction in the myristoylation of
C. albicans ARF, which is consistent with
fungistatic activity (9). Recent elucidation of the S. cerevisiae and C. albicans NMT crystal structures (10-12) will facilitate a better understanding of the inhibition mechanisms of these enzymes.
Parasitic kinetoplastid protozoa of the species Leishmania
and Trypanosoma are causative agents of some of the most
debilitating tropical infections currently affecting world populations
(see, on the World Wide Web, www.who.int/tdr/index.html). Metabolic labeling studies have identified at least ten
[3H]myristate-labeled proteins in Leishmania
major, including the ARF-like protein 1 (53). and the infective
stage specific, hydrophilic acylated surface proteins (HASPs) (13, 14).
HASPB, a candidate vaccine for visceral leishmaniasis (15), requires
N-terminal myristoylation and palmitoylation for translocation to the
parasite plasma membrane (16). Myristate is also a component of the
glycophosphatidylinositol (GPI) lipid anchors that tether the major
classes of surface molecules in trypanosomatid parasites. In
Leishmania, the myristate-containing GPI anchors of the
surface glycoinositolphospholipids are remodeled during synthesis by a
reaction involving myristate exchange from a myristoyl-CoA donor (17).
In mammalian stages of Trypanosoma brucei, fatty acid
remodeling is also important in synthesis of the variant surface
glycoprotein GPI anchor, which contains two myristates in its
diacylglycerol domain (18). As a consequence, myristate analogues are
toxic in T. brucei, probably due to a cumulative effect on
GPI anchor, phospholipid, and N-myristoylated protein
synthesis (19, 20).
Here, we show that myristate analogues (nonspecific inhibitors of
NMT) are also toxic to Leishmania and describe the molecular characterization of NMT in both L. major and
T. brucei. As with the pathogenic fungi,
expression of NMT is essential for viability in these parasites,
whereas modulation of enzyme activity inhibits parasite growth,
yielding aberrant phenotypes, presumably due to disrupted modification
of "downstream" target proteins.
Leishmania Culture and Inhibitor Assays--
L. major
(MHOM/IL/81/Friedlin) promastigotes were maintained in vitro
at 26 °C as described (13). Logarithmically growing parasites at
~8 × 106 ml PCR Amplification, Subcloning, and DNA Analysis--
A 677-bp
fragment of the NMT gene was amplified from L. major genomic DNA, using Taq DNA polymerase (Promega)
at 55 °C annealing temperature and the primers, NMTfor
(5'-GAGATYAACTTYCTSTGCGTSCAC-3') and NMTrev
(5'-RCCRTCRCCIIIRCCRAACTT-3'), based on the NMT "signature sequences" (EINFLCVH and KFGXGDG, available on the World
Wide Web at www.expasy.org/prosite/) and adjusted to L. major codon bias. The amplified fragment, nmt, was
cloned into pGEM-T (Promega) and used to screen a gridded L. major Friedlin genomic cLHYG cosmid library (21). Of the 21 positive clones identified, 12 mapped to the same contiguous sequence
on chromosome 33. L. major NMT was subcloned from cosmid
L8010 (21) into pBS SK+, as 1.6- and 2.0-kb
EcoRI/SacI fragments.
The same degenerate primers were used to amplify and sequence portions
of the NMT genes from Leishmania donovani
(MHOM/ET/67/L28; accession number AY072732), Leishmania
infantum (MHOM/FR/78/LEM75; accession number AY072731), and
Leishmania mexicana (MNYC/BZ/62/M379; accession number
AY072730). The T. brucei NMT gene was identified as
TRYP10.0.001826-6, part of a 15-kb contig on chromosome 10, in data
deposited at www.sanger.ac.uk/cgi-bin/nph-proj-omniblast.html. A
175-amino acid fragment of the Trypanosoma cruzi NMT is also available (accession number AI069625), spanning residues 193-371 of
the T. brucei open reading frame (ORF) (Fig. 2).
Bacterial Expression Constructs--
The L. major NMT
ORF was amplified from genomic DNA, using primers ORFfor
(5'-TATGGATCCATATGTCTCGCAATCCATCGAACTCCG-3') and ORFrev
(5'-ATAGGATCCATCGATCTACAGCATCACCAAGGCAACCTG-3'). The L. major HASPA ORF was amplified from plasmid pXS (14) using primers Hfor (5'-AGATATACCATGGGAAGCTCTTGCACGAAGGAC-3') and Hrev
(5'-ATAACTCGAGCTAGTTGCCGGCAGCGTGCT-3'). The amplified fragments
were cloned into pGEM-T, and the sequences of both fragments were
verified as wild type.
For expression in Escherichia coli, the cloned NMT ORF was
digested with NdeI/BamHI and subcloned into the
expression vector, pET15b (Novagen), to obtain plasmid pNMT. The HASPA
ORF was digested with NcoI/XhoI and cloned into
the expression vector pET28a (Novagen), generating plasmid pHASPA. The
recombinant HASPA expressed from this plasmid has an N-terminal
myristoylation motif (MGSSCTK).
Production of Antibodies and Immunoblotting--
Expression of
N-terminally His-tagged recombinant L. major NMT (from pNMT)
was induced by isopropyl-1-thio-
For immunoblotting, wild type parasites were lysed in SDS gel-loading
buffer, proteins were separated by SDS-PAGE, and the resulting blots
were probed with purified NMT antiserum (abSK805; 1:2000 dilution),
anti-HASPB (14), anti-BiP (22), anti-GP63 (polyclonal antibody, 1:2000;
gift of Robert McMaster), anti-SHERP (1:5000) (23), and anti-CAP5.5
(monoclonal antibody, 1:100) (24). Immune complexes were detected using
an ECL kit (Amersham Biosciences).
Functional Co-expression of Recombinant L. major NMT in E. coli--
This assay was based on the co-expression system described
in Ref. 2. E. coli BL21(DE3)pLysS cells were
cotransformed with pNMT and pHASPA, either singly or in combination.
2-ml cultures were grown to A600 0.5 in LB
media containing ampicillin, kanamycin, and chloramphenicol, and
expression of recombinant proteins was induced by the addition of 1 mM isopropyl-1-thio- Parasite Membrane Fractionation--
5 × 108
midlog phase parasites were lysed and separated into membrane and
cytoplasmic fractions as described (23). Proteins in both fractions
were analyzed by SDS-PAGE and immunoblotting, as described above.
Targeted Deletion/Complementation of the L. major NMT
Gene--
To target both NMT alleles, plasmids were generated
containing either a hygromycin resistance gene (hyg) or a
puromycin resistance gene (pac) flanked with DNA sequences
immediately upstream and downstream of the NMT ORF. These constructs,
based on the pX63-HYG vector (25), were designated pNMT-HYG and
pNMT-PAC, respectively. To generate pNMT-HYG, a 873-bp fragment
immediately upstream of the NMT ORF was amplified using primers NKO1F
(ATAGCaagcttGCGACGCGTGGGACTGTGACA) and NKO1R
(GTGCATgtcgacCGCAGTATTCGTGTGCGTGT). Cloning sites are shown in
lowercase, and L. major sequence is shown in uppercase. The
amplified product was digested with HindIII/SalI
and cloned into pX63-HYG, generating plasmid pXN-HYG. Subsequently, a
1110-bp fragment immediately downstream of the NMT ORF was amplified
using the primers NKO2F (ATCGTcccgggTTTACGCNCCGCCCCACCTTTCCG)
and NKO2R (CATGCTagatctGCACCAGTGCTCGGCGGTCGGC) and cloned into
the SmaI/BglII sites of pXN-HYG, generating
pNMT-HYG. To construct pNMT-PAC, the pac gene was released
from plasmid plmcpb-PAC (26) as previously described (27) and used to
replace the hyg gene in pNMT-HYG, following
SpeI/BamHI digestion. Linear targeting regions
(produced by digesting pNMT-HYG with SapI/MscI
and pNMT-PAC with SapI/NcoI) were purified and
used sequentially to transfect midlog phase L. major
promastigotes as described (28). Transfected parasites were plated in
the presence of 32 µg ml
For complementation of transgenic L. major or overexpression
in wild type parasites, pNMT was digested with
ClaI/SmaI prior to cloning the insertion into the
expression vector pTEX (29), generating pTEX NMT NEO. Parasites were
electroporated with 20 µg of plasmid DNA, prior to plating in the
presence of 20 µg ml RNAi in T. brucei--
A 543-bp fragment within the
NMT ORF was amplified from T. brucei genomic DNA using the
primers Tb-NMT-For (5'-ACTCCAATAATGGTTCTAGAGAAGAGAGA-3') and Tb-NMT-Rev (5'-TCGTCTTTTtctagaCCCACCG-3'). T. brucei sequence is shown in uppercase, and cloning sites are
underlined. The PCR fragment was digested with XbaI and
cloned into the XbaI sites of the RNA interference
(RNAi) vector, p2T7Ti, which supports expression of
double-stranded RNA from tetracycline-inducible T7 promoters (30),
generating p2T7-NMT.
The procyclic T. brucei strain 29-13 (31), containing the
genes expressing T7 RNA polymerase and tetracycline repressor, was
maintained in vitro at 27 °C in SDM-79 medium
supplemented with 10% fetal calf serum, 25 µg ml
The bloodstream T. brucei strain 90-13 (31) was maintained
in vitro at 37 °C with 5% CO2 in HMI-9
medium supplemented with 10% fetal calf serum, 5 µg
ml Electron Microscopy--
Parasites were washed three times in
serum-free M199 medium and resuspended at 1 × 107
ml Effect of Myristoyl-CoA Analogues on the Growth of L. major--
Since myristate analogues are nonspecific
competitive inhibitors of NMT, we first tested these compounds for
their effect on Leishmania growth and division in culture.
Specifically, the myristate analogues 2-hydroxymyristate and
4-oxatetradecanoate were incubated with procyclic L. major
over a 72-h time course (Fig. 1). Whereas
the untreated parasites continued to divide, growth in the
inhibitor-treated cultures slowed after 24 h, and viable parasite
numbers reached a plateau by 48 h, depending on the inhibitor
concentration. At 48 h, there was >50% reduction in parasite
numbers compared with untreated cells, with both inhibitors showing a
dose-dependent effect. On microscopic examination, the inhibitor-treated cells showed extensive morphological differences (rounded cells with variable motility and intracellular organelle distention) when compared with the untreated parasites (data not shown).
These results demonstrate that, as with T. brucei, myristate
analogues are toxic to Leishmania promastigotes, most likely due to global effects on GPI anchor and phospholipid biosynthesis as
well as N-myristoylation of target proteins. It was not
possible to correlate the loss of viability observed in the presence of these analogues with reduced N-myristoylation of specific
targets, since the single well characterized Leishmania
N-myristoylated protein, HASPB, is only expressed later in the
growth cycle, following differentiation of procyclic parasites to
infective metacyclics (13, 14).
Cloning and Characterization of Leishmania
N-Myristoyltransferase--
The L. major NMT gene was
cloned by degenerate PCR amplification of genomic DNA followed by
library screening. Contiguous sequence (3480 bp) generated from the
subcloned gene fragments contained a 1266-bp ORF, coding for a 48.5-kDa
protein of pI 6.1 (accession number AF305956). This deduced protein
shares identity with NMT from other species, as shown in the sequence
alignment in Fig. 2.
L. major NMT shares 42, 41, 43, 44, and 40% overall amino
acid identity with Homo sapiens, S. cerevisiae,
C. albicans, C. neoformans, and
Plasmodium falciparum NMTs, respectively. The NMTs of other
Leishmania species (L. donovani, L. infantum, L. mexicana) are highly conserved with the
L. major enzyme (97, 96, and 96%, respectively, in
the region between the signature sequences (data not shown)), whereas
in the related trypanosomatid species, T. brucei, amino acid
identity through the ORF drops to 54% (Fig. 2). Similar analysis of a
T. cruzi NMT fragment (spanning residues 193-371 of the
T. brucei ORF) shows 44% identity with L. major NMT.
As with all NMTs so far characterized, the L. major and T. brucei enzymes are divergent at their N termini (which are not involved in catalysis) and do not contain N-terminal lysine-rich regions (that have been implicated in ribosomal targeting of human and
mouse NMTs) (34). Conversely, many of the key fungal NMT residues
predicted to be directly involved in the enzyme mechanism or to have
critical regulatory roles (6) are conserved in L. major and
T. brucei. An exception is Cys217 of S. cerevisiae NMT (and conserved in other fungal species); mutation
of this residue has a selective effect on the S. cerevisiae NMT peptide binding site (35). In L. major and T. brucei NMTs, this residue is replaced by Gly. High resolution
structural analysis of the C. albicans enzyme has revealed
that two negatively charged residues, Glu173 and
Leu451, form the floor of the active site pocket of the
enzyme (10). These residues are conserved in both L. major
and T. brucei NMTs, but there is also an insertion of ~20
amino acids, rich in Lys and Glu, close to the first of these "pocket
floor" residues in the parasite enzymes (Fig. 2). An additional
insertion of 29 amino acids is found further downstream in T. brucei NMT. These features suggest that the parasite NMTs might
form distinctive secondary and tertiary structures that could affect
their activities relative to the human and fungal enzymes.
L. major NMT Is a Constitutively Expressed Single Copy
Gene--
L. major NMT is a single copy gene (as determined
by endonuclease digestion, blotting, and hybridization; data not
shown). Similarly, the genomes of the New World species, L. amazonensis, and T. brucei also contain single
NMT genes.
Leishmania parasites cycle between extracellular
promastigotes (within the sandfly vector) and intracellular amastigotes
(principally within macrophages of the mammalian host). To investigate
NMT expression during the L. major life cycle, equivalent
numbers of noninfective (logarithmic or procyclic) and infective
(metacyclic) promastigotes and amastigotes (extracted from
BALB/c mouse lesions) were lysed and analyzed by SDS-PAGE. After
blotting, NMT was immunodetected as a single 48.5-kDa protein, present
in all life cycle stages (Fig.
3A). In the same samples,
expression of HASPB, a member of the hydrophilic acylated surface
protein family, was restricted to infective parasite stages as
previously demonstrated (13, 14). Expression of the constitutive
endoplasmic reticulum marker protein, BiP, was used as a control
for equivalent loading. Thus, NMT is constitutively expressed in
L. major.
Subcellular Localization of L. major NMT--
NMT has been
localized to the cytoplasm in S. cerevisiae and does not
appear to be associated with cellular membranes (36). Conversely, a
fraction of Drosophila NMT has been shown to be membrane-bound (37), and NMTs in mammalian cells (38, 39) are all
partially associated with membranes, as peripheral membrane proteins.
This is also the case for L. major NMT (Fig. 3B).
Following separation of parasite extracts into membrane and cytoplasmic fractions, immunoblotting with anti-NMT revealed that >70% of the
L. major enzyme is associated with membranes. As controls for these experiments, GP63 (the major GPI-anchored surface protein of
Leishmania species) was detected solely in the membrane
fraction, as expected, whereas SHERP, a hydrophilic protein that
fractionates with membranes only after in vivo cross-linking
(23), was detected only in the cytoplasmic fraction (Fig.
3B).
Functional Analysis of L. major NMT in E. coli--
A bacterial
co-expression assay system (2) was modified to demonstrate the activity
of L. major NMT in vivo. In these experiments, a
plasmid expressing L. major NMT (pNMT) and a plasmid
expressing the ORF of an NMT substrate were co-transformed into
E. coli. Protein expression was induced with
isopropyl-1-thio-
Following induction of protein expression in the co-transformed
bacteria, proteins migrating at 48.5 and 17 kDa were specifically expressed in whole cell lysates (Fig. 4, upper
panel, track 6). These were identified
as NMT and HASPA, respectively, by immunoblotting (data not shown).
Both proteins were radiolabeled when [3H]myristate was
included in the incubation (Fig. 4, lower panel, track 6). The apo-NMT is known to form a high
affinity complex with myristoyl-CoA (5), explaining the presence of the
radiolabeled 48.5-kDa NMT. The HASPA (migrating at 17 kDa) was
myristoylated by the recombinant NMT expressed from pNMT. This labeling
is resistant to treatment with 1 M hydroxylamine,
indicative of an amide linkage, characteristic of
N-myristoylation (data not shown). Transformation with
pHASPA alone (Fig. 4, lower panel,
track 4) generated no detectable radiolabeled
product, despite high levels of protein expression (compare
tracks 4 and 6). Expression of NMT in
the absence of plasmid-encoded substrate resulted in myristoylation of
an endogenous 25-kDa E. coli protein (Fig. 4,
lower panel, track 2).
Similar results have been observed previously (40, 41). Thus, under
these assay conditions, recombinant L. major NMT is active
in vivo and is required for the N-myristoylation of HASPA.
Leishmania NMT Encodes an Essential Enzyme in Extracellular
Parasites--
To investigate the importance of NMT expression in the
Leishmania life cycle, gene targeting was used to generate
transgenic parasites lacking one or both copies of the NMT
gene. Linear constructs, containing hygromycin (hyg) or
puromycin (pac) resistance genes flanked by sequences from
the dihydrofolate reductase locus for constitutive expression (42),
were targeted to the NMT locus, using 5' and 3' intergenic regions for
homologous recombination (Fig.
5A). In these experiments,
heterozygous lines (containing integrated hyg or
pac genes) were readily obtained: of 10 clones analyzed for
each integration; six hyg clones and five pac
clones resulted from precise chromosomal NMT replacement.
However, after a second round of targeting, although it was possible to
select clones that were resistant to both drugs, none of these were
NMT chromosomal nulls, despite the analysis of >30 clones
by immunoblotting and 20 of these clones by Southern blotting (data not
shown). It was only possible to generate true NMT chromosomal nulls
when episomal copies of the NMT gene were introduced into
heterozygous parasites, prior to targeting the second NMT
allele for deletion. Of 17 clones analyzed after complementation in
this way, seven had lost both NMT chromosomal copies.
Analysis of a series of typical transgenic clones is shown in Fig.
5B. Integration of the hyg or/and pac
gene constructs (Fig. 5A) into the NMT locus is
detected on 13.6- and 12.5-kb SalI fragments, respectively,
in transgenic clones 2 (+/
The expression levels of NMT in the four transgenic clones described
above were analyzed (Fig. 5C). Using BiP as a control protein, the equivalent NMT expression levels as compared with wild
type were 0.4-, 5.9-, and 6.5-fold for transgenic clones 2-4,
respectively. Thus, loss of one NMT allele results in ~60% decreased
protein expression, whereas parasites containing the episome express
considerably enhanced levels of NMT, due to the increased gene copy
number on the plasmid. Growth of the four clones was also monitored
over a 6-day period (Fig. 6D).
Transgenic clones 2-4 showed a decrease in growth rate compared with
wild type, although total parasite numbers were comparable after 6 days
in culture.
Overall, these data resemble those obtained by others when trying to
remove sequentially both copies of genes encoding essential proteins in
Leishmania (e.g. Refs. 43-45). As a consequence,
we conclude that NMT is an essential gene for
Leishmania promastigote viability.
Overexpression of NMT Is Lethal in Leishmania--
To further
investigate the effects of increased NMT expression on parasite
viability, the pTEX NEO NMT episome was transfected into wild type
L. major. The resulting transgenic parasites, selected by
their resistance to neomycin, did not differentiate into metacyclics and developed abnormal morphology during growth on plates and in liquid
culture. These cells were predominantly rounded, with disrupted
subcellular organization and disintegration of nuclear membranes (Fig.
6, C-E; compare with wild type parasites in Fig. 6,
A and B). They contained increased numbers of
lysosomes and, as a dominant feature, previously undescribed oval
structures, which appeared to be bound by membranes. Staining with
malachite green (Fig. 6E) confirmed that these
structures are rich in lipids.
Immunoblotting of transfected parasite lysates (prepared from pTEX NEO
NMT cells collected at the same time as those processed for electron
microscopy) showed a 10.5-fold increase in the expression of NMT
protein compared with the wild-type control (Fig. 6F). All
clones overexpressing NMT maintained viability for a short time, but
cell death occurred after 1-2 weeks in all cases, compared with clones
transfected with the pTEX vector alone (Fig. 6G). This is a
more extreme phenotype than that seen in Fig. 5D, in which
transgenic parasites with a modest 5-6-fold increase in NMT expression
showed wild type morphology but had a growth defect. Hence, whereas a
5-fold increase in NMT expression is tolerated by
Leishmania, an increase to >10-fold is lethal. Increasing
the drug selection pressure on +/ Expression of the T. brucei NMT Gene Is Essential for Viability in
Procyclic and Bloodstream Trypanosomes--
Whereas methods for
regulated expression of transgenes are still under development in
Leishmania, RNAi has been established as a
reproducible technique for gene down-regulation and phenotypic analysis
in T. brucei (30, 46, 47). Thus, to determine the phenotype
of trypanosomes deficient in NMT, RNAi constructs were generated to target expression from the T. brucei NMT gene
in both vector (procyclic) and mammalian (bloodstream) parasite stages, following induction with tetracycline.
Growth of procyclic trypanosomes of the parental clone, 29-13 (carrying
integrated copies of the T7 RNA polymerase and tetracycline repressor
genes) and the transgenic clone, p2T7/NMT, were monitored over a time
course (Fig. 7A). Both induced
and noninduced p2T7/NMT parasites grew at similar rates for the first 3 days, when the cultures were split. From that point, up until 9 days
postinduction, growth of the induced p2T7/NMT clone was negligible,
whereas the noninduced control replicated as before. Throughout this
period, the parental parasites, 29-13, showed a similar (if more rapid) growth pattern to the noninduced control. When cells were split 1 day
earlier (at 2 days postinoculation), cell death still occurred at day 4 (data not shown).
Immunoblotting parasite lysates collected at intervals following
tetracycline induction demonstrated a decrease in NMT protein expression by 72 h postinduction, correlating with the time at which the cultures were split (Fig. 7B). No further NMT
expression could be detected by 96 h, by which time there was a
significant reduction in parasite growth and increase in cell death
(Fig. 7A). This effect was still apparent at 9 days
postinduction, with very low parasite viability and no detectable NMT
protein. From 96 h postinduction, there was an accumulation of
parasites with aberrant morphology, including rounded or abnormally
elongated cells, carrying more than one flagellum and displaying
partial plasma membrane detachment (Fig. 7C and data not
shown). This array of phenotypes could be attributed to the effects of
NMT depletion on modification of a range of downstream
N-myristoylated target proteins. Expression of one of these,
the procyclic-specific, cytoskeleton-associated protein CAP5.5 (24),
was unperturbed during the time course of this experiment (Fig.
7B).
Similar results were obtained when growth of bloodstream trypanosomes
of the parental (90-13) and transgenic (Bp2T7/NMT) clones were compared
(Fig. 7D). The induced parasites grew normally for 72 h
and then began to round up, leading to a high rate of cell death by
96 h postinduction. Therefore, we can conclude that, as in
L. major, NMT protein is essential for viability of T. brucei in both life cycle stages of the parasite.
The importance of N-myristoylation in the synthesis of
functional proteins, often with a role in signaling or other
fundamental cellular processes, has focused interest on the enzyme that
catalyzes this process, myristoyl-CoA:protein
N-myristoyltransferase. The development of peptide and
peptidomimetic anti-NMT reagents for potential therapeutic use against
fungal diseases has been aided by knowledge of the key structural
features of the enzyme. Current published data suggest that both
specific and sensitive inhibition of NMT activity can be achieved when
compared with host enzyme activity (8, 12). Against this background, we
have cloned and characterized NMT from several trypanosomatid parasite
species, in order to evaluate the functional importance of
N-myristoylation in these organisms and to investigate the
potential of NMT as a drug target in the diseases that they cause.
The L. major and T. brucei NMTs share high
similarity with other characterized NMTs in their primary sequence,
including conservation of key amino acids essential for catalysis,
including the "pocket floor" residues identified at the active site
(10) and the hydrophobic residues (Phe170 and
Leu171 in S. cerevisiae) critical for
myristoyl-CoA binding and product release (11). The noncatalytic N
termini of the parasite enzymes show sequence divergence and do not
contain the polylysine domains implicated in ribosomal targeting,
regions that are also missing from the NMTs of other lower eukaryotic
species studied to date. In this context, the recent report of post-
rather than co-translational N-myristoylation of the
proapoptotic protein, BID, following caspase-8 cleavage, suggests that
NMT is localized to more than one distinct intracellular site (48), at
least in mammalian species. This observation might correlate with the
recent demonstration of a second mammalian NMT cDNA, possibly
arising from a second gene, and the expression of different NMT
isoforms in mammalian cells (49). Leishmania NMT, encoded by
a single copy gene, is unequally distributed between membranes and the
cytosol (Fig. 3).
NMT is constitutively expressed in Leishmania species and
T. brucei, as in the pathogenic fungi. Expression of
L. major NMT in E. coli has provided a robust
functional assay for enzyme activity in vivo, as originally
demonstrated with S. cerevisiae NMT, which has fatty acid
and peptide specificities indistinguishable from those of the native
enzyme when expressed in bacteria (41). As with S. cerevisiae NMT, two major radiolabeled species are detected in
E. coli expressing Leishmania NMT and a suitable
substrate: the acyl-enzyme complex and the myristoylated substrate.
Both of these are still produced, although less efficiently, when the parasite-specific 20-amino acid NMT domain is deleted (data not shown),
most likely due to altered higher order structure of the protein.
Consistent with this interpretation, construction of a homology model
of L. major NMT based on the S. cerevisiae
structure has shown that this insertion is on the surface of the
protein and does not affect the active site (50). More detailed
in vitro analyses with purified soluble enzyme will
delineate the importance of this and other parasite-specific regions
and residues.
The fungicidal and trypanocidal properties of myristate analogues,
nonspecific inhibitors of NMT, have been well documented (7, 20, 51),
although these compounds show distinct organism-specific differences.
The two compounds tested here clearly inhibit the growth of L. major promastigotes, presumably due to cumulative effects on
various cellular processes. Competitive peptide binding inhibitors will
be required to verify this effect and correlate growth inhibition with
reduced NMT activity. Using an alternative molecular genetic approach,
we have confirmed that NMT expression is essential for the viability of
both Leishmania promastigotes and T. brucei
procyclic/bloodstream form parasites, whereas increased NMT protein
levels also have deleterious effects. In particular, the accumulation
of lipid bodies in Leishmania NMT overexpressors, prior to
cell death, may be a phenotype specific to cells that express high
levels of myristate-containing, GPI-anchored surface molecules. By
comparison, 5-6-fold overexpression of NMT activity in S. cerevisiae has no effect on growth kinetics or cell morphology (3). Aberrant N-myristoylation of substrates probably
contributes to this unusual parasite phenotype, at the expense of other
metabolic fates for the available myristate, which include GPI anchor
remodeling (17). However, the recent demonstration of de
novo myristate synthesis in T. brucei (52)
suggests that fatty acid synthetic pathways may also exist in
Leishmania and that myristate concentration may not be a
limiting factor contributing to the accumulation of lipid bodies.
Further experiments are under way to assess the importance of NMT
expression in intracellular amastigotes of Leishmania and to
determine the downstream targets that contribute to the loss of
viability observed. It is unlikely that nonmyristoylation of the HASP
proteins results in cell death, since transgenic parasites lacking HASP
function are as viable as wild type cells (27). By contrast, reduced
ARF myristoylation has been shown to cause growth arrest in C. albicans and S. cerevisiae (8, 9), suggesting that a
member of this protein family may be important for viability in
trypanosomatid parasites. Whatever the myristoylated proteins involved,
the distinctive features of trypanosomatid NMTs, including their single
gene copy number, conserved sequence, and expression in
mammalian-infective parasite stages, suggest that these enzymes may be
appropriate targets for the development of antiparasitic drugs.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-subunit of many
signal-transducing, heteromeric G proteins. Although some myristoylated
proteins are cytosolic, many are associated with cellular membranes
where myristoylation facilitates membrane attachment. The addition of
myristate can also stabilize protein-protein interactions, and many
acylated proteins require this modification for full expression of
their biological function (2). Genetic studies have shown that the
NMT gene is essential for the survival of
Saccharomyces cerevisiae (3) and the pathogenic fungi,
Candida albicans and Cryptococcus neoformans (4).
Given the critical role of N-myristoylation in the cell, NMT
has been developed as a target for the development of antifungal
chemotherapeutic agents.
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DISCUSSION
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1 were incubated with
different inhibitors (dissolved in 100% ethanol as 100 mM
stocks) that had previously been conjugated to fat-free bovine serum
albumin as described (16). Ethanol was added to noninhibitor-treated
cells to the same final concentration (1%). At the time points
indicated, aliquots were removed in triplicate for counting; all motile
organisms were scored, including those with abnormal morphology. Each
time course was done in triplicate, and the whole experiment was
repeated three times.
-D-galactopyranoside in E. coli BL21(DE3)pLysS, and the cells were subsequently
lysed in 6 M Gu-HCl, prior to affinity chromatography using
Ni2+-nitrilotriacetic acid-agarose (Qiagen). Eluted protein
was further purified by SDS-PAGE and used for immunization and
generation of rabbit polyclonal antisera (Eurogentec).
-D-galactopyranoside. Immediately, the cultures were divided into two 1-ml aliquots, with 150 µCi of [9,10-3H]myristate (Amersham
Biosciences) in 10 µl of ethanol added to one aliquot and 10 µl of
ethanol alone added to the second. The cells were then incubated at
37 °C for a further 3 h, prior to centrifugation,
phosphate-buffered saline washing, lysis, and analysis by SDS-PAGE.
Radiolabeled proteins were detected by autoradiography at
80 °C
using Hyperfilm MP (Amersham Biosciences).
1 hygromycin and 20 µg
ml
1 puromycin, clones were amplified, and DNA/protein was
extracted for analysis as described (27). The DNA probes HYG
(1027 bp) and PAC (600 bp) were generated by digesting
pNMT-HYG and pNMT-PUR with SpeI/BamHI. An
NMT N-terminal probe (986 bp) was produced by digesting
NMT-pTEX with XhoI/SalI. Immunoblotted proteins
were detected as described above.
1 G418, amplification, and analysis
as described above and in Ref. 27.
1
hygromycin, and 25 µg ml
1 G418. Midlog phase parasites
were electroporated with 20 µg of NotI-linearized p2T7-NMT
DNA, as described (32). The parasites were allowed to recover
overnight, and transfectants were selected using 2.5 µg
ml
1 phleomycin and grown for 3 weeks until stable cell
lines had been established. The expression of double-stranded RNA was
induced by the addition of 1 µg ml
1 tetracycline to
parasites diluted to 1 × 106 ml
1 in
SDM-79 medium. Cell numbers were monitored using a Beckman Coulter
counter. Protein samples were prepared at 24-h intervals and analyzed
by immunoblotting, as described above.
1 hygromycin, and 2.5 µg ml
1 G418.
Midlog phase parasites were electroporated with 10 µg of NotI-linearized p2T7-NMT DNA, as described (33). The
parasites were allowed to recover for 6 h, before the addition of
2.5 µg ml
1 phleomycin. The cells were transferred to
24-well culture plates and grown for 10 days until stable cell lines
had been established. Expression of double-stranded RNA was induced by
the addition of 1 µg ml
1 tetracycline to parasites
diluted to 2 × 104 ml
1 in HMI-9 medium.
Cell numbers were monitored as above.
1. Parasites were fixed in 4% paraformaldehyde and
2.5% glutaraldehyde in phosphate-buffered saline, pelleted, fixed
again in 1% osmium tetroxide in sodium cacodylate buffer, treated with
1% tannic acid, dehydrated through an increasing ethanol series
(staining en bloc in 2% uranyl acetate at the 30% ethanol stage),
infiltrated, and embedded in TAAB 812 resin (TAAB Laboratories
Equipment Ltd.). 60-nm ultrathin sections, cut on a Leica UCT
ultramicrotome, were contrasted with uranyl acetate and lead citrate
and examined on a Philips CM100 transmission electron microscope. For
lipid staining, malachite green was added at 0.5% to the primary
fixative solution just prior to fixing on ice for 2 h. Uranyl
acetate was used as a light counterstain at the 30% ethanol stage
during dehydration, and final contrasting with uranyl acetate and lead
citrate, as described above, was omitted.
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View larger version (14K):
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Fig. 1.
Myristate analogues inhibit logarithmic
growth in L. major. Parasites were grown in
triplicate wells at 26 °C and counted at intervals, either untreated
( ) or following the addition of the inhibitors 4-oxatetradecanoate
(
, 1 mM;
, 0.2 mM) and 2-hydroxymyristate
(
, 1 mM;
, 0.2 mM). Viable parasite
numbers were counted in triplicate, and S.E. values were determined.
The data shown represent one of three independent experiments.
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Fig. 2.
Alignment of L. major and
T. brucei N-myristoyltransferases with the
NMTs from other species. The deduced open reading frames of
L. major NMT (AF305956I) and T. brucei NMT
(TRYP10.0.001826-6) are aligned with NMTs from other species, using the
ClustalW multiple sequence alignment program (available on the
World Wide Web at www.ebi.ac.uk/clustalw): C. albicans
(CANAL, P30418), S. cerevisiae
(SCEREV, P14743), C. neoformans
(CRYNEO, P34809), P. falciparum
(PFALC, AF206306), and H. sapiens
(HUMAN, P30419). The positions of the conserved signature
sequences used in the design of forward and reverse primers for
amplification of the nmt fragment from L. major
genomic DNA are indicated by the arrows; the ~20-amino
acid insertion regions of the L. major (residues 137-157)
and T. brucei (residues 132-156) NMTs are
underlined. Boxed amino acids are key residues
involved in myristoyl-CoA binding (solid lines)
or peptide binding (dashed lines) in yeast
species. Filled circles identify the pocket floor
residues, Glu173 and Leu451 in C. albicans.
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Fig. 3.
Leishmania NMT is
membrane-associated and constitutively expressed during the parasite
life cycle. A, total parasite lysates from procyclic
(P), metacyclic (M), and amastigote
(A) stages of L. major (1 × 107
parasites/track) were size-separated and immunoblotted with anti-NMT,
anti-HASPB, or anti-BiP (to monitor equivalent protein loading between
tracks). B, lysed procyclic parasites were fractionated into
membrane (M) and cytoplasmic (C) fractions, prior
to size separation and immunoblotting with anti-NMT, anti-GP63, and
anti-SHERP.
-D-galactopyranoside, and
N-myristoylation was monitored by radiolabeling. A number of
Leishmania substrates have been used in these assays,
including ARL-1 (data not shown) and HASPA, a second member of the
hydrophilic acylated surface protein family. In the experiment in Fig.
4, E. coli were co-transformed
with pHASPA and pNMT prior to metabolic labeling with
[3H]myristate. The products were then separated by
SDS-PAGE, followed by Coomassie Blue staining and autoradiography.
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Fig. 4.
Recombinant NMT myristoylates a
Leishmania substrate protein in E. coli. pNMT and pHASPA were expressed in E. coli, either separately or together, as recombinant proteins of
48.5 and 17 kDa, respectively (arrowheads in top
panel) in the presence of [3H]myristate,
following induction with
isopropyl-1-thio- -D-galactopyranoside (tracks
2, 4, and 6). The radiolabeled
products (the NMT-myristoyl-CoA binary complex and myristoylated HASPA)
were detected by autoradiography (lower panel),
followed by densitometry to determine relative incorporation levels.
The data shown represent one experiment that was repeated three times.
Coomassie Blue-stained, SDS-PAGE-separated proteins are shown in the
upper panel.
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Fig. 5.
The NMT gene encodes an
essential enzyme in Leishmania promastigotes.
A, restriction maps of the wild type (diploid)
NMT locus and targeted single alleles containing insertions
of either the hyg or pac gene. The positions of
5' and 3' dihydrofolate reductase flanking regions are shown as
cross-hatched boxes; hatched boxes
show the intergenic regions used for gene targeting. B,
BamHI; M, MscI; N,
NcoI; P, PstI; S,
SalI; Sp, SapI. B, Southern
blot analysis of parasite DNAs from wild type (+/+) (lane
1); single allele hyg replacement
(+/ NMT::HYG) (lane 2); complemented
single allele hyg replacement (+/
NMT::HYG
[pTEX NEO NMT]) (lane 3); complemented double
allele hyg/pac replacement
(
NMT::HYG/
NMT::PAC [pTEX NEO NMT])
(lane 4). DNAs were digested with SalI
(HYG, PAC, and NEO blots) or
PstI (NMT blot) prior to size separation,
transfer, and hybridization with HYG, PAC,
NEO, or NMT probes. Size markers (kb) are shown
on the right of each blot. C, protein extracts
from the four wild type and transgenic clones shown in B
(5 × 106 parasites/track) were immunoblotted with
anti-NMT and anti-BiP. Blots were scanned, and the NMT expression
levels were determined by densitometry relative to the NMT/BiP
expression ratio in wild type parasites. D, growth of the
four parasite lines shown in B, monitored over 6 days. Shown
are wild type (+/+;
); single allele hyg replacement
(+/
NMT::HYG;
); complemented single allele
hyg replacement (+/
NMT::HYG [pTEX NEO NMT];
); and complemented double allele hyg/pac replacement
(
NMT::HYG/
NMT::PAC [pTEX NEO NMT];
×).
NMT::HYG), 3 (+/
NMT::HYG [pTEX NEO NMT]), and 4 (
NMT::HYG/
NMT::PAC [pTEX NEO NMT]). In transgenic clones 3 and 4, introduction of the NMT episome (pTEX NEO
NMT) is detected by hybridization of a NEO probe to SalI
fragments of 6 kb (linearized plasmid) and 12 kb (probable plasmid
dimer). The second chromosomal NMT gene has been removed by
gene targeting in clone 4 (
NMT::HYG/
NMT::PAC [pTEX NEO NMT]), in
which multiple NMT copies are detectable as episomal 6-kb
PstI fragments, whereas the 3-kb chromosomal alleles are missing.
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Fig. 6.
Overexpression of Leishmania
NMT is lethal in promastigotes. Wild type L. major and transgenic parasites overexpressing NMT (+/+ [pTEX NEO
NMT]) were sectioned and viewed by transmission electron microscopy.
A and B, wild type parasites viewed in
longitudinal and transverse section, respectively; C-E,
transverse sections of pTEX NEO NMT transgenics; the white
arrowheads indicate oval structures only seen in parasites
overexpressing NMT. E, counterstaining with malachite green
demonstrates that these structures are rich in lipids. F,
immunoblotted protein extracts from wild type (wt) and
overexpressing parasites (+/+ [pTEX NEO NMT]) (5 × 106/track), probed with anti-NMT and anti-BiP. Blots were
scanned, and relative NMT expression levels were determined as in Fig.
5C. G, the survival of eight independent clones
each of +/+ [pTEX] ( ) and +/+ [pTEX NEO NMT] (
) was monitored
up to 14 days in culture. Scale bar in
A-E, 500 nm; n, nucleus; k,
kinetoplast; fp, flagellar pocket.
NMT::HYG [pTEX NEO NMT]
parasites (transgenic clone 3 in Fig. 5) by 10-fold resulted in
increased NMT expression and similar morphological changes to those
shown in Fig. 6, C-E.
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Fig. 7.
The NMT gene encodes an
essential enzyme in procyclic and bloodstream stages of T. brucei. A, growth of the T. brucei procyclic
parental line, 29-13 (expressing T7 RNA polymerase and Tet repressor,
) and the transfected line, p2T7/NMT, in the presence (
) and
absence (
) of tetracycline, monitored over a 9-day time course.
B, immunoblotted protein extracts from the parental line,
29-13 (without Tet) and the p2T7/NMT line at 0-216 h after induction
with tetracycline, probed with anti-NMT, anti-BiP, and anti-CAP5.5.
1 × 107 parasites were lysed and analyzed in each
track. C, phase-contrast images of noninduced
(NI) and 4-day postinduction (I) procyclics.
Scale bar, 15 µm. D, growth of the
T. brucei bloodstream parental line, 90-13 (
) and the
transfected line, Bp2T7/NMT, in the presence (
) and absence (
) of
tetracycline, monitored over a 5-day time course.
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DISCUSSION
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge contributions from the following colleagues: Jeremy Mottram, John Kelly, George Cross, and Doug LaCount for parasite strains and vectors; Al Ivens for help with cosmid library screening; the T. brucei and T. cruzi genome projects for access to sequence data; Jay Bangs, Robert McMaster, and Keith Gull for antibodies; Claire Allen and Belinda Hall for assistance with T. brucei transfection; and Kate Brown, Liz Carpenter, Robin Leatherbarrow, Philip Nugent, and members of the Smith laboratory for discussion.
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FOOTNOTES |
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* This work was supported by Wellcome Trust Grants 045493/Z/95/Z and 061343/Z/00/Z.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors have contributed equally to this work.
§ Recipient of an Overseas Research Scholarship. Present address: Division of Virology, National Institute for Medical Research, London NW7 1AA, United Kingdom.
¶ Recipient of a research studentship from the UK Biological and Biotechnological Scientific Research Council.
Present address: Dept. of Biological Sciences, Lancaster
University, Lancashire LA1 4YQ, United Kingdom.
** To whom correspondence should be addressed. Tel.: 44-20-75945282; Fax: 44-20-75945283; E-mail: d.smith@ic.ac.uk.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M211391200
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ABBREVIATIONS |
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The abbreviations used are: NMT, N-myristoyltransferase; HASP, hydrophilic acylated surface protein; GPI, glycophosphatidylinositol; ORF, open reading frame; RNAi, RNA interference; ARF, ADP-ribosylation factor; contig, group of overlapping clones.
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