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The Shikimate Pathway and Its Branches in Apicomplexan Parasites
Craig W. Roberts,1 Fiona Roberts,2 Russell E. Lyons,1
Michael J. Kirisits,5,7 Ernest J Mui,5,7 John Finnerty,8
Jennifer J. Johnson,5,7 David J. P. Ferguson,4
John R. Coggins,3 Tino Krell,3 Graham H. Coombs,3
Wilbur K. Milhous,10 Dennis E. Kyle,10 Saul Tzipori,9
John Barnwell,12 John B. Dame,13 Jane Carlton,11,a
and Rima McLeod5,6,7,b
1Department of Immunology, University of Strathclyde, 2Department
of Pathology, Victoria Infirmary, and 3Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow, and 4Nuffield Department of
Pathology, John Radcliffe Hospital, Oxford University, Oxford,
United Kingdom; 5Departments of Ophthalmology and Visual
Sciences and 6Departments of Medicine and Pathology and
Committees on Genetics and Immunology, University of Chicago, and7Department of Medicine, Michael Reese Hospital, Chicago, Illinois;
8Department of Biology, Boston University, and 9Tufts University
School of Veterinary Medicine, Boston, Massachusetts; 10Division of
Experimental Therapeutics, Walter Reed Army Institute of Research
and 11National Institutes of Health, Bethesda, Maryland;12Department of Parasitic Diseases, Centers for Disease Control
and Prevention, Chamblee, Georgia; 13University of Florida,
School of Veterinary Medicine, Gainesville
The shikimate pathway is essential for production of a plethora of aromatic compounds in
plants, bacteria, and fungi. Seven enzymes of the shikimate pathway catalyze sequential con-
version of erythrose 4-phosphate and phosphoenol pyruvate to chorismate. Chorismate is then
used as a substrate for other pathways that culminate in production of folates, ubiquinone,
napthoquinones, and the aromatic amino acids tryptophan, phenylalanine, and tyrosine. The
shikimate pathway is absent from animals and present in the apicomplexan parasites Toxo-
plasma gondii, Plasmodium falciparum, and Cryptosporidium parvum. Inhibition of the pathway
by glyphosate is effective in controlling growth of these parasites. These findings emphasize the
potential benefits of developing additional effective inhibitors of the shikimate pathway. Such
inhibitors may function as broad-spectrum antimicrobial agents that are effective against bac-
terial and fungal pathogens and apicomplexan parasites.
Overview of the Shikimate Pathway and Its Branches,
Derivatives, and Organization
Characterization of the shikimate pathway of apicomplexan
parasites is of special interest because the pathway is absent
from animals but apparently essential for survival of the para-
sites [1, 2]. Thus, understanding the shikimate pathway in these
organisms may assist in the development of new antiparasitic
agents. The plethora of information available on the shikimate
pathway of other organisms (bacteria, fungi, and plants) provides
a starting point for these studies. However, existing data reveal
differences in shikimate pathways of bacteria, fungi, and plants
and suggest many possible modes of regulation and inhibition of
the pathway. It remains to be seen whether the pathway in apicom-
plexan parasites provides yet more diversity.
The shikimate pathway operates in the cytosol of bacteria
and fungi, but in plants it is also known to operate in plastid
organelles. The pathway utilizes phosphoenol pyruvate and
erythrose 4-phosphate to produce chorismate through seven
catalytic steps [3, 4] (figure 1). It is a pathway with multiple
branches. While branches exist from various intermediate pro-
ducts of the pathway, chorismate represents the major branch
point, and various branches give rise to many end products.
Derivatives of chorismate include tryptophan via an anthranilate
intermediate [3, 4], phenylalanine and tyrosine via prephenate or
arogenate [3–5], and vitamin K and metal chelators containing
dihydroxybenzoic acid, such as enterochelin, via isochorismate
[3, 4]. Chorismate also is used for production of ubiquinone and
p-aminobenzoic acid (PABA), which is converted into folates.
The shikimate pathway also gives rise to many secondary metabo-
The Journal of Infectious Diseases 2002;185(Suppl 1):S25–36q 2002 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2002/18504S-0004$02.00
Presented in part: Fourth International Toxoplasmosis meeting,Drymmen, Scotland, 26–30 July 1996; Fifth International Toxoplasmosismeeting, Marshall, California, 1–6 May 1999; Keystone Symposium onOpportunistic Infections in AIDS, Keystone Colorado, 2–8 April 1998;Woods Hole Molecular Parasitology meeting, Woods Hole, Massachusetts,September 1999; Joint Meeting of the American Society of Parasitologistsand the Society of Nematologists, Monterey, California, 6–9 July 1999.
Financial support: NIH (AI-44328); Welcome Trust; Research to PreventBlindness Foundation; Fulbright Scholar Awards; British PathologySociety Traveling Fellowship; Howard Hughes Foundation; World HealthOrganization; Brennan, Blackmon, Hoffman, and Koshland families; Toxo-plasmosis Research Institute.
a Current affiliation: Parasite Genomics Group, Institute for GenomicResearch, Rockville, Maryland.
Reprints or correspondence: Dr. Rima McLeod, University of Chicago,
Visual Sciences Center, AMBH S206, 5841 South Maryland, Chicago, IL
60637 ([email protected]).
S25
Figure 1. The shikimate pathway and major branches from chorismate. A, The shikimate pathway catalyses the conversion of phosphoenoly py-ruvate and erythrose 4-phosphate to chorismate by 7 enzymatic steps (boxed). Major branches from chorismate include ubiquinone synthesis, whichis dependent on 8 enzymes (boxed) (B); folate synthesis, which is dependent on 6 enzymes (boxed) (C); tryptophan synthesis, which is dependent on7 enzymes (boxed) (D); phenylalanine and tyrosine synthesis, which occur by separate pathways that branch from prephenate and involve the actionof a further 5 enzymes (boxed) (E). Further branches exist from chorismate in certain organisms, including isoprenoid synthesis, and from otherintermediates, such as quinate synthesis from dehydroquinate. There is currently direct evidence only for the branch leading to folates in apicomplexanparasites. * There are many variations and subtleties in tyrosine and phenylalamine synthesis in different species.
lites, including flavanones and napthoquinones [6], and there is an
alternative version of the pathway with aminated intermediates
that give rise to aminohydroxybenzoate (AHBA), the precursor of
the ansamycin antibiotics [7]. Our recent work on apicomplexans
has demonstrated a role for the shikimate pathway in the pro-
duction of PABA and folates in these organisms [1].
Twenty percent of the carbon derived from carbohydrate cat-
abolism is utilized in the shikimate pathway in certain organisms
[8]; therefore, it is a major part of their metabolism. Animals lack
the shikimate pathway enzymes and, with the exception of dihy-
drofolate reductase (DHFR), lack enzymes downstream of chor-
ismate. These observations suggested to us that this pathway
and its branches in apicomplexan parasites might provide a num-
ber of interrelated targets for antiparasitic agents [1].
The shikimate pathway is a viable herbicide target [9]. Speci-
fically, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase,
the sixth enzyme in the pathway, is the target for glyphosate, a
widely used herbicide (Roundup; Sigma) [3, 4, 9]. The com-
pound also inhibits EPSP synthase of Toxoplasma gondii and
the growth of T. gondii, Plasmodium falciparum, and Crypto-
sporidium parvum [1]. A number of other compounds that inhibit
various other enzymes in the shikimate pathway have been
described, and some inhibit the growth of bacteria [10]. It
is surprising that none of these has been developed commercially.
Two of these compounds, the fluorinated analogues of shikimate
(6R)-6-fluoroshikimate and (6S)-6-fluoroshikimate, inhibit the
growth of P. falciparum in vitro, presumably through competi-
tive inhibition of the shikimate pathway [2].
Genes encoding many shikimate pathway enzymes have been
cloned from various bacteria, plants, and fungi. In contrast, choris-
mate synthase genes of T. gondii and P. falciparum are the only
apicomplexan representatives that have been cloned and se-
quenced [1]. In addition, we have recently obtained evidence for
the presence of chorismate synthase in Eimeria bovis, Plasmo-
dium vivax, and C. parvum (unpublished data).
Our recent demonstration that the shikimate pathway is im-
portant for the survival of a number of apicomplexans has raised
a number of interesting questions that we and others are currently
addressing. First, since the shikimate pathway is generally oper-
ative in the plastid of plants [3, 4], we are interested in whether
some or all of it occurs in the plastid-like organelle of apicom-
plexans. Second, we are interested in how the apicomplexan shi-
kimate pathway is regulated, particularly as this may provide
clues regarding the best potential targets for new antimicrobial
agents. Third,what is the importance of branches of the shikimate
pathway for various stages of the life cycles of apicomplexan
parasites, including latent stages?
The Need for New Antimicrobial Agents for Treatment
of Apicomplexan Parasites
The phylum Apicomplexa consists of a large number of proto-
zoa, including species of Toxoplasma, Plasmodium, Eimeria,
Theileria, Cryptosporidium, and Babesia. These parasites are re-
sponsible for a variety of diseases in humans and livestock. For
example, T. gondii, a very common parasite, causes ocular and
neurologic lesions and can result in systemic disease and death
when acquired in utero [11]. It has been estimated to cost more
than two billion dollars per annum to care for children born with
congenital infection in the United States [12]. Moreover, toxo-
plasmosis is a devastating disease in those who are immunocom-
promised due to AIDS, chemotherapy, malignancy, transplan-
tation, or autoimmune diseases and the involved therapies [11].
The most effective chemotherapy for toxoplasmosis currently
comprises the combination of the antifolate agents pyrimeth-
amine and sulfadiazine. These are both associated with significant
adverse reactions. Pyrimethamine inhibits the action of DHFR
and thus the production of tetrahydrofolate, which is required
for a number of reactions. Since DHFR is present in humans,
pyrimethamine-induced depletion of folate cofactors can lead to
bone marrow suppression. Sulfadiazine can cause allergy and is
commonly poorly tolerated, which results in problems with com-
pliance. Furthermore, this combination therapy acts only on the
rapidly dividing tachyzoite stage associated with active disease
and does not affect the quiescent bradyzoite stage that forms
cysts throughout the body. Thus the encysted bradyzoite forms
remain a reservoir of infection even in treated individuals and
can give rise to repeated disease reactivation. This is especially
evident in congenitally infected and immunocompromised indi-
viduals. Disease reactivation is common in the eye and brain,
where the consequences can be serious. Therapy is often required
on multiple occasions and for long periods of time [11].
Malaria is caused by the apicomplexan parasites of the genus
Plasmodium. The four common species of Plasmodium (P. falci-
parum, P. malariae, P. vivax, and P. ovale) infect millions of
people and account for the death of more than 2 million children
annually. The emergence of drug resistance has limited the use-
ful life span of many antimalarial medicines. Current treatments
include inhibitors, such as pyrimethamine and sulfadoxine, of
the folate synthetic pathway [13].
C. parvum is another apicomplexan parasite that causes dis-
ease in humans and livestock. It causes debilitating and life-
threatening diarrheal illness in patients infected with human
immunodeficiency virus. C. parvum infection can also occur in
epidemics, in which it is most severe in the young and elderly.
There is currently no effective treatment for the disease caused
by this parasite [14, 15].
An advantage of developing agents that target the shikimate
pathway is their potential to inhibit the growth of bacterial
and fungal pathogens and apicomplexan parasites. There are
growing concerns associated with the emergence of multidrug-
resistant bacteria, such as Staphylococcus aureus, as well as a
number of enterococci, Streptococcus pneumoniae, and enteric
gram-negative bacteria [16]. In addition, there is now consider-
able worry about the increased occurrence of multidrug resis-
tance among Mycobacterium tuberculosis, Salmonella typhi, Shi-
Shikimate Pathway Branches in Apicomplexan ParasitesJID 2002;185 (Suppl 1) S27
gella species, and Neisseria gonorrhoeae [17]. Furthermore, in
immunocompromised patients, multiple infections with fungi
and a variety of different classes of organisms, including myco-
bacteria and apicomplexan parasites, may occur. There is thus
great need for the development of new antimicrobial agents, and
one that targeted several of these diseases would be especially
beneficial.
Evidence for the Shikimate Pathway
in Apicomplexan Parasites
Chemotherapeutic, biochemical (figures 2, 3), and genetic
(figure 4) evidence has been obtained for shikimate pathway
enzymes in apicomplexan parasites, including T. gondii, P.
falciparum, and C. parvum [1]. In addition, we have completed
sequencing the P. vivax aroC encoding chorismate synthase and
included it in the multisequence alignment in figure 4 (GenBank
accession number AF451277).
The first evidence we obtained for the presence of the shiki-
mate pathway in T. gondii was the ability of glyphosate to restrict
growth of T. gondii in vitro (figures 2A, 3). As this inhibition was
overcome by the addition of PABA to treated cultures, it is likely
that the shikimate pathway is important for supply of folate pre-
cursors in this parasite. Additional control experiments demon-
strated that the addition of PABA could reverse the inhibitory
effect of sulfadiazine. Sulfadiazine inhibits the action of dihy-
Figure 2. Functional and enzymatic evidence for the shikimate pathway in Toxoplasma gondii. A, Glyphosate inhibition of T. gondii tachyzoitegrowth in human foreskin fibroblasts (HFF) in vitro as demonstrated by [3H]uracil uptake by the parasite (3.12 mM glyphosate vs. none,P , :002). RH, T. gondii tachyzoites of the RH strain; P/S, pyrimethamine and sulfadiazine. Glyphosate toxicity for a variety of cell lines, includingHFF (dividing cells), bovine kidney cells (MDBK), and erythrocytes were assessed. The concentrations used were not toxic. B, p-aminobenzoic acid(PABA) rescue of glyphosate growth inhibition. Added PABA reversed the effects of glyphosate and sulfadiazine (S) but not pyrimethamine (P). C,Inhibition of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase enzyme activity by 1 mM glyphosate. Parasite extracts were produced at 4�C. D,In vivo activity of glyphosate and pyrimethamine against T. gondii. ND4 mice were infected intraperitoneally with T. gondii tachyzoites (RH strain)and immediately treated orally with glyphosate alone (100 mg/kg/day; no surviving mice by day 9), with pyrimethamine alone (12.5 mg/kg/day;10% surviving mice by day 9), or with these agents combined at the specified concentrations, or they were left untreated (0% survival at 9 days).Treatment of mice with either drug alone was not sufficient to prevent death (i.e., was similar to that of control mice). In contrast, 50% of mice admi-nistered glyphosate and pyrimethamine simultaneously survived infection (at 7 days after infection, P , :05). Results were similar in 2 replicateexperiments; the data shown are a composite of these experiments (10–12 mice/group). PABA and folate were administered ad libitum in the diet.Reprinted with permission from [1].
Roberts et al.S28 JID 2002;185 (Suppl 1)
dropteroate synthase and thus the incorporation of PABA into
the folate biosynthetic pathway. As expected, however, addition
of PABA to pyrimethamine-treated cultures had no antagonistic
effect on efficacy of pyrimethamine. This is because pyrimeth-
amine exerts its effect later in the pathway by inhibiting dihydro-
folate reductase (figure 2B). Confirmation of a T. gondii EPSP
synthase, which can be inhibited by glyphosate, was obtained
by measuring enzyme activity in crude T. gondii extracts
(figure 2C): 1 mM glyphosate inhibited 85% of the EPSP
synthase activity.
Ability of glyphosate to inhibit growth in vitro of asexual
erythrocytic forms of P. falciparum also was tested. At a concen-
tration of �1 mM, glyphosate completely inhibited parasite
growth, an effect that could be abrogated by treatment with
PABA or folate (figure 3A). Similarly, growth in vitro of
C. parvum was curtailed by glyphosate (figure 3B), although in
this case, addition of PABA to cultures did not abrogate inhi-
bition (data not shown). This last observation suggests that the
shikimate pathway is absolutely essential in this parasite, pre-
sumably providing precursors for biochemical pathways other
than the one that leads to folates. Alternatively, a lack of effect
from added PABA may simply reflect poor transport of the com-
pound into C. parvum. The ability of glyphosate to act in synergy
with sulfadiazine and pyrimethamine, which interfere with fo-
late synthesis by inhibiting dihydropteroate synthase and DHFR,
respectively, was tested in vitro. Glyphosate appeared to act
synergistically with both of these compounds in inhibiting in
vitro growth of T. gondii (table 1).
Glyphosate has had extensive toxicological testing and has
been defined as “practically non-toxic.” As such, it requires in
excess of 2000 mg/kg to cause acute toxicity in mammals [18].
Thus the effect of glyphosate on apicomplexan parasites was
assessed by use of a murine model of toxoplasmosis. These ex-
periments found that although glyphosate had no effect when
administered alone, it significantly reduced mortality when
given in combination with low doses of pyrimethamine (figure
2D). This demonstrates that inhibitors of EPSP synthase can act
in synergy with conventional antifolates and may be a useful
addition to the agents used against apicomplexan parasites.
This approach may have great benefit in treating drug-resistant
malaria as we also found that glyphosate is equally effective
in vitro against both pyrimethamine-sensitive and pyrimeth-
amine-resistant P. falciparum lines.
More recently, (6R) and (6S)-6-fluoroshikimate have each
been shown to inhibit the growth of P. falciparum in vitro [2].
The effect of (6R)-6-fluoroshikimate was ablated by the addition
of aromatic amino acids (phenylalanine, tyrosine, tryptophan),
PABA, and para-hydroxybenzoate (PHBA). In contrast, addi-
tion of aromatic amino acids and PHBA or of aromatic amino
acids alone was not sufficient to ablate the effects of the (6R)-6-
fluoroshikimate. The effect of PABA alone was not tested in
these experiments, making it difficult to determine if PHBA
and the aromatic amino acids are necessary to reverse the effects
of this inhibitor [2].
EPSP synthase activity is detectable in crude extracts of
T. gondii, and low levels of chorismate synthase can also be
measured in crude T. gondii extracts by use of an anaerobic
assay [1, 19]. Further studies detected 3-dehydroquinate dehy-
dratase and shikimate kinase in T. gondii lysates (3.0 and
1.6 mU/mg protein, respectively). 3-dehydroquinate dehydra-
Figure 3. Evidence for the shikimate pathway in Plasmodium falci-parum and Cryptosporidium parvum. A, Functional evidence for theshikimate pathway in P. falciparum. Glyphosate inhibition of in vitrogrowth of asexual erythrocytic forms and p-aminobenzoic acid(PABA) and folate (FA) antagonism of growth inhibition. Glyphosate(1.08 mM ) completely inhibited asexual stages of the D6 clone invitro, and both PABA and FA antagonized the inhibitory effect of gly-phosate. D6 is a clone from the African Sierra I/UNC isolate, which issensitive to chloroquine and pyrimethamine. PABA was �1000-foldmore effective than FA. B, Glyphosate inhibition of C. parvum growth.Glyphosate and paromomycin (PRM) each inhibited parasite growth(P , :001). None of the concentrations of glyphosate were toxic to themonolayer. “Media” indicates that inhibitor was not added. Reprintedwith permission from [1].
Shikimate Pathway Branches in Apicomplexan ParasitesJID 2002;185 (Suppl 1) S29
Figure 4. Comparison of deduced amino acid sequences of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Synechocystis,Escherichia coli, and Solanum chorismate synthases. The function(s) of the insertions, which have no counterparts in the genes of any otherorganisms, including Synechocystis species, Solanum lycospersicum, Saccharomyces cerevisiae, Neurospora crassa, Hemophilus influenzae, orStaphylococcus aureus, remains to be determined. Pink indicates 100% identity; deep blue indicates 60% homology; light blue indicates conserva-tive substitution of residues.
tase was partially purified and shown to be heat labile, indicating
that it is similar to the type I dehydroquinate dehydratases found
in higher plants [20]. Previous studies using crude extract of
P. falciparum detected shikimate kinase (4 mU/mg protein),
3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase
(4 mU/mg protein), and shikimate dehydrogenase (90 mU/mg
protein) [21]. Thus, there is now evidence for the presence in
apicomplexan parasites of all of the shikimate pathway enzymes
except 3-dehydroquinate synthase.
Chorismate synthase genes of 2 apicomplexan parasites,
T. gondii and P. falciparum, have been identified and sequenced
(GenBank accession numbers U 93689 and AF008549, respec-
tively), and the sequence of P. vivax was recently completed
(GenBank accession number AF451277). The deduced amino
acid sequences share all the amino acids known to be highly
conserved and have considerable identity with each other and
the chorismate synthases from other species. However, both
the T. gondii and P. falciparum proteins differ from other
known chorismate synthases in possessing a number of unique
insertions (figure 4). We have also recently identified homolo-
gous genes in C. parvum and E. bovis by Southern blotting
with a probe derived from the T. gondii chorismate synthase
cDNA (Johnson J, Roberts CW, McLeod R; unpublished data).
In addition to cloning and sequencing the T. gondii and
P. falciparum chorismate synthase genes, using cDNA, we
have completed the sequencing of the T. gondii genomic aroC
(chorismate synthase) gene and have identified the P. falciparum
genomic aroC sequence on chromosome 11 in the ongoing
P. falciparum genome project (Kirisits M, Roberts CW, McLeod
R, et al.; unpublished data). The T. gondii gene has many introns,
but the P. falciparum sequence has none (Kirisits M, Roberts
CW, McLeod R, et al.; unpublished data) [22]. Of interest, the 50
non-coding region of T. gondii aroC contains a potential GCN4
binding motif (TGACTC). The possible significance of this is
discussed below in the section on Regulation of the Shikimate
Pathway. We have determined that the T. gondii chorismate
synthase gene (aroC) is single copy, which makes it amenable
to gene deletion experiments, which are in progress.
Phylogeny and Subcellular Location of the Shikimate
Pathway in Apicomplexan Parasites
Presence of an apicomplexan algal plastid-like organelle
[23–25] and nuclear-encoded enzymes translocated and func-
tioning in plant plastids (reviewed in [26]) initially provided a
conceptual basis for our work [27, 28]. Phylogenetic analysis of
the T. gondii and P. falciparum chorismate synthases, using
parsimony and distance [28] and maximum likelihood [29] anal-
yses, indicated a puzzling monophylogeny of apicomplexan and
fungal enzymes, whereas other apicomplexan nuclear genes
have been located in the clade with alveolata, which in turn is
alignedwithciliatesandplants [30],andplastidgenesandnuclear
genes targetedtotheplastidhavebeenalignedwithcyanobacteria
and algae [23, 31–33]. Thus, these phylogenetic analyses do not
provide information that is useful for determining subcellular
localization of the pathway. Fitzpatrick et al. [22] interpreted
their immunofluorescencedataasdemonstratingcytosolic locali-
zation of P. falciparum chorismate synthase. Definitive data are
needed to accurately characterize phylogeny and subcellular
localization of each of the shikimate pathway enzymes.
Potential Branches Derived from the Shikimate
Pathway in Apicomplexans
In plants, bacteria, and fungi, chorismate is used as a substrate
for the production of many end products, including ubiquinone
and the aromatic amino acids tryptophan, tyrosine, and phenyl-
alanine. The possible role of the shikimate pathway in supplying
chorismate for the synthesis of ubiquinone and aromatic amino
acids in apicomplexan parasites remains to be confirmed. It has
been demonstrated that treatment of human fibroblasts with
interferon-g induces degradation of tryptophan and thus pre-
vents the growth of intracellular T. gondii tachyzoites [34, 35].
Moreover, transfection of T. gondii tachyzoites with the
Escherichia coli trpB gene, which encodes the b subunit of
tryptophan synthase, has been shown in the presence of exoge-
nous indole to confer resistance to this effect of interferon-g
[36]. Thus, T. gondii tachyzoites would appear to be dependent
on exogenous tryptophan supplied through their host cell and
to be incapable of synthesizing this aromatic amino acid. The
Table 1. Effects of glyphosate alone and together with antimicro-bial agents that inhibit folate synthesis on replication of Toxoplasmagondii.
Experiment no.,
treatment
[3H]uracil
incorporation,
cpma
Predicted
[3H]uracil
incorporation,
cpm, for A + Bb
Ratio,
actual:predictedc
Experiment 1
Untreated 71,449 ^ 3763
Glyphosate (drug A) 28,138 ^ 2216
Sulfadiazine (drug B) 25,026 ^ 4365
Drug A + drug B 2368 ^ 418 9856 0.24
Experiment 2
Untreated 64,343 ^ 1222
Glyphosate (drug A) 25,097 ^ 1398
Pyrimethamine (drug B) 69,217 ^ 3253
Drug A + drug B 9354 ^ 2126 25,097 0.37
NOTE. Concentrations of 3.125 mM glyphosate, 6.25mg/mL21 sulfadiaz-
ine, and 0.025mg/mL21 pyrimethamine were used. These results are representa-
tive of at least 2 replicate experiments. Adapted with permission from [1].a [3H]uracil indicates incorporation of labeled tritiated uracil into T. gondii
nucleic acid; data are mean ^ SEM of a minimum of 3 replicate wells.b Predicted [3H]uracil incorporation for drug A plus drug B, if effect is only
additive not synergistic, is calculated as (cpm drug A £ cpm drug BÞ/ ðcpm of
untreated culture).c A ratio of actual:predicted of ,1 is considered synergistic; a ratio of
actual:predicted <1 is considered additive.
Shikimate Pathway Branches in Apicomplexan ParasitesJID 2002;185 (Suppl 1) S31
bradyzoites of T. gondii have not been studied in this respect, and
it is possible that they may be unable to scavenge tryptophan. In
contrast to T. gondii tachyzoites, it has been reported that trypto-
phan is not essential for the survival of P. falciparum, although
tyrosine is an essential aromatic amino acid [37]. These two
amino acids are synthesized from chorismate but by divergent
enzymatic pathways (figure 1). These observations suggest that
certain apicomplexan parasites have independently lost differ-
ent biochemical pathways from chorismate. These differences
may reflect the various evolutionary pressures operative in phy-
logenetically related parasites living in diverse environments.
Regulation of the Shikimate Pathway
There is no published information on regulation of the shiki-
mate pathway in apicomplexan parasites, but information from
the study of bacteria, fungi, and plants provides clues to how
this might occur. Understanding the regulation of the shikimate
pathway in apicomplexans may furnish valuable information
that will underpin therapeutic intervention.
Regulation of the shikimate pathway is complex and varies
greatly between organisms. The best-studied model is E. coli,
in which the shikimate pathway is regulated by controlling the
abundance and activity of DAHP synthase. Regulation occurs
at the protein level by feedback inhibition and at the transcrip-
tional level by repression [38–49]. Feedback inhibition is the
major controlling factor of these two mechanisms [38]. In Sac-
charomyces cerevisiae, aromatic amino acid biosynthesis is
under the “general control” mechanism, which enables the
organism to respond to limited amino acid supply. Thus, the
expression of genes encoding shikimate pathway enzymes aro3
and aro4, which encode DAHP synthase-Phe and -Tyr, respec-
tively, and aro1, which encodes the pentafunctional arom protein
(which catalyses steps 2–6 in the pathway), are regulated by the
control-activator protein GCN4. This protein binds to a specific
upstream promoter element (TGACTC) present in the genes it
regulates, thereby controlling their expression [50–59].
Starvation for amino acids also leads to increased synthesis of
shikimate pathway enzymes in the plant Arabidopsis thaliana
[60]. This finding shows that plants are capable of cross-pathway
metabolic regulation; however, in plants the regulation of the
pathway is complicated by the subcellular localization of the
enzymes. The bulk of the shikimate pathway enzyme activities
are located in the plastids [61], but there is also evidence for
cytosolic isoenzymes [62]. In a number of plants, such as carrot
(Dauchus carota) and A. thaliana, two isoforms of DAHP
synthase have been identified [63, 64]. For example, in tobacco
there is a plastidic Mn2+-stimulated isoform of DAHP synthase
and a Co2+-stimulated cytosolic isoform [65]. The genes encod-
ing the plastidic form of DAHP synthase in potatoes, tobacco,
tomato, and Arabidopsis species have been isolated and charac-
terized [4]. Induced expression of the plastidic form of the
enzyme has been demonstrated by wounding or pathogen attack
of potato tubers [4] and elicitor treatment or light exposure of
parsley cells [66] resulting in the accumulation of metabolites
of the shikimate pathway. Evidence suggests this induced
expression is due to increases in the levels of transcription of
the specific genes since cycloheximide and actinomycin D both
inhibit this induction [67].
Two DAHP synthase isoforms are differentially expressed in
A. thaliana, in which only one isoform (DHS1) was up-regulated
during wounding or pathogenic attack, while the other form
remained stable [64]. In tomato (Lycopersicon esculentum), two
distinct genes encoding plastidic forms of DAHP synthase have
been identified, and each has different expression levels in vari-
ous organs [68]. Other examples of tissue-specific expression
include EPSP synthase in petunia [69] and isozymes of choris-
mate synthase in tomato [70].
In the photosynthetic protozoon Euglena gracilis, two forms
of EPSP synthase have been identified. One is a monofunctional
60-kDa enzyme that appears to be localized to the chloroplast
[71], and the other is a domain of the multifunctional 165-kDa
AROM protein, which appears to be cytosolic [72]. These two
proteins are differentially expressed according to environmental
conditions, with the AROM predominant in dark-grown cells but
down-regulated in light-grown cells and with the monofunc-
tional plastid-associated form undetectable in dark-grown cells
but up-regulated by light [73].
The Shikimate Pathway and Its Branches
as Antimicrobial Agent Targets
The presence of the shikimate pathway in a large number of
pathogenic organisms, including bacteria, fungi, and apicom-
plexan parasites, and its absence from mammals make it an
attractive target for the development of antimicrobial agents.
Each enzyme in the pathway provides a potential target, and if
any of the branches (figure 1) are operative in appropriate life-
cycle stages, then this potential chemotherapeutic exploitation
is amplified even more. Subcellular organelle transit and trans-
location signals (the enzymes that cleave signal sequences) and
regulatory mechanisms for the pathway also may offer potential
for exploitation as targets of antimicrobial agents.
The shikimate pathway has already been exploited by the
commercial herbicide glyphosate, an inhibitor of EPSP syn-
thase. The presence of this pathway in a wide range of patho-
genic organisms, including bacteria, fungi, and apicomplexan
parasites, emphasizes the potential of targeting the pathway
in these organisms. Glyphosate inhibits the growth of both
gram-negative and -positive bacteria in vitro [74–77]. Certain
bacterial chorismate synthases, DHFRs, and chorismate mutases
also have been shown to be essential [1, 4].
Although relatively high concentrations of glyphosate are
required to restrict the growth of the apicomplexan parasites
Roberts et al.S32 JID 2002;185 (Suppl 1)
tested so far, the results provide impetus for the development of
new anti-shikimate pathway therapies. The relatively high con-
centration of glyphosate required raises the important question
of whether it is possible to alter the structure of glyphosate to
make better inhibitors. Extensive testing of glyphosate analogues
[78] has not provided compounds that are as effective as glypho-
sate for the inhibition of EPSP synthase. These empiric findings
ledMousdaleetal. [78] tosuggest thatglyphosatemaybeaunique
“toxophore” (i.e., that it may not be possible to improve upon the
way this small and simple molecule interacts with EPSP syn-
thase). The findings of McRobert and McConkey [79] suggest
that inhibition of chorismate synthase is lethal for P. falciparum.
The ultimate goal of inhibiting the shikimate pathway is assisted
by a number of studies that have resulted in the development of
an array of compounds, including various substrate analogues,
capable of interfering with various steps in this pathway [2].
Most of these inhibitory activities have been demonstrated by
use of assays using recombinant or purified enzymes or crude
homogenates of organisms; however, some inhibitors have been
assessed by their ability to inhibit the pathogen in vitro or in vivo.
A range of compounds inhibit EPSP synthase activity, such as
various analogues of its substrate, shikimate 3-phosphate [80–
83]. The role of these compounds as potential antimicrobials
has not been fully examined even though many of the com-
pounds were found to be active at low micromolar or even nano-
molar levels. Other enzymes in the shikimate pathway are also
amenable to inhibition. The fluorine-containing analogue of
phosphoenol pyruvate acts as a substrate for DAHP synthase,
the first enzymatic step in the shikimate pathway [84]. The maxi-
mum rate at which DAHP synthase can convert this competitive
inhibitor to the fluorine-containing analogue of DAHP is only
about 1% that for phosphoenol pyruvate. Similarly, substrate
analogues of DAHP are competitive inhibitors of dehydroqui-
nate synthase, the second enzymatic step [85, 86].
Substrate analogues of other shikimate pathway enzymes also
inhibit the shikimate pathway. Some of these analogues do not
inhibit their immediate enzyme, but are converted to substrate
analogues for subsequent enzymes in the pathway. For example,
in E. coli, (6S)-6-fluoroshikimate and (6R)-6-fluoroshikimate
are substrates for shikimate kinase and are converted to (6S)-
6-fluoro-EPSP and (6R)-6-fluoro-EPSP, respectively [10]. Where-
as (6R)-6-fluoro-EPSP is an inhibitor of chorismate synthase,
(6S)-6-fluoro-EPSP is slowly converted by chorismate syn-
thase to form 6-fluorochorismate [87, 88]. This 6-fluorochoris-
mate would appear to inhibit 4-aminobenzoic acid synthesis
and account for the ability of this compound to inhibit E. coli
in vitro [10, 89]. Of interest, 6-fluorochorismate can inhibit
chorismate synthase of Neurospora crassa [90]. The difference
observed between the E. coli and N. crassa systems may be due
to the bifunctional nature of the N. crassa enzyme and may
suggest that 6-fluoroshikimate interferes with the flavin reduc-
tase activity present on the N. crassa enzyme.
(6S)-6-fluoroshikimate protects mice from intraperitoneal
infection with E. coli, Pseudomonas aeruginosa, and S. aureus;
however, spontaneous resistance occurred at a high frequency
to this compound, preventing its development for clinical use
[10]. More recently, (6R) and (6S)-6-fluoroshikimic acid have
each been shown to inhibit the growth of P. falciparum in vitro
(IC50 of 1:5 £ 1025 and 2:7 £ 1024 M, respectively) [2]. The rela-
tively low IC50s of these compounds against P. falciparum high-
light the potential of rationally designed substrate analogues of
this pathway in the treatment of disease caused by apicomplexan
parasites.
Development of Resistance to Inhibitors
A potential concern for all new drugs is the development of
drug resistance. Although glyphosate tolerance has not been a
problem in the field, it is possible to artificially produce glypho-
sate tolerance in plants and bacteria. Glyphosate-tolerant plants
have been obtained by selecting tolerant cell lines while increas-
ing the concentration of glyphosate in growth media [91].
Glyphosate-tolerant bacterial cell lines have been produced by
mutagenesis [92]. These organisms have made it possible to
study glyphosate resistance, an important biological mechanism
with implications in medicine and agriculture.
Two different mechanisms to overcome EPSP synthase inhi-
bition by glyphosate have been identified. The first of these is
the production of glyphosate-tolerant mutant EPSP synthase. In
Salmonella typhimurium, a single amino acid substitution of
proline to serine at amino acid position 101 in EPSP synthase is
sufficient to confer resistance to glyphosate, and it is transferable
to E. coli via a cosmid [93]. In Klebsiella pneumoniae, substi-
tution of glycine-96 with alanine in EPSP synthase reduces sen-
sitivity to glyphosate [94].
The second mechanism of resistance is the production of ele-
vated levels of EPSP synthase activity. For example, in the
higher plant Corydalis sempervirens, glyphosate-resistant cells
have a 40-fold increase in EPSP synthase activity, while levels
of the other shikimate pathway enzymes remain the same as in
wild-type plants [95]. Similarly in a glyphosate-tolerant Petunia
hybridoma cell line, EPSP synthase activity is increased 15- to
20-fold [96]. This increase in activity may be the result of
increased gene expression, as in C. sempervirens [97], or by
gene amplification, as has been demonstrated in petunia [98],
carrot [99], and tobacco [100]. While the adaptive mechanism
appears to be the more common in plants and fungi, studies of
glyphosate resistance in E. gracilis have demonstrated that
either mechanism may occur [101].
Resistance to fluoroshikimate occurs spontaneously in E. coli.
This resistance correlates with a reduced ability to take up
shikimate and has been mapped at or near shiA, which is one of
at least two loci that determine shikimate uptake in E. coli
[102]. Other shikimate-like inhibitors might suffer from the
Shikimate Pathway Branches in Apicomplexan ParasitesJID 2002;185 (Suppl 1) S33
same problems, making altering modes of entry or “non-shiki-
mate”–like inhibitors more promising strategies.
Conclusions
Characterization of the shikimate pathway in apicomplexan
parasites may provide opportunities for the development of
new antimicrobial agents. The inhibitor studies that use glypho-
sate demonstrate empirically that inhibition of EPSP synthase,
and thus the shikimate pathway, is an effective means of inhibit-
ing the growth of T. gondii, P. falciparum, and C. parvum. In a
similar manner, the ability of (6R)-6-fluoroshikimate to inhibit
P. falciparum suggests that inhibition of chorismate synthesis is
another effective means for controlling the growth of at least
P. falciparum. Increasing our understanding of the shikimate
pathway in apicomplexans may not be only of biologic inter-
est—it may expedite the discovery and design of new anti-
microbial agents.
Many opportunistic pathogens, such as Pneumocystis carinii
[103], M. tuberculosis [104], C. parvum, and T. gondii simulta-
neously infect AIDS patients and rely on the shikimate pathway.
It is therefore possible that a single compound that inhibits the
shikimate pathway or combinations of synergistic compounds
that inhibit various enzyme targets in this and interrelated path-
ways [58, 105] could simultaneously eliminate multiple patho-
gens infecting a patient. Thus, they could reduce or eliminate
the problems of antimicrobial agent resistance.
A recent description of bacterial genomics and targets in the
shikimate pathway highlights and discusses the enzymes in the
shikimate and other pathways that are particularly promising tar-
gets [105]. For apicomplexans, to date, EPSP synthase, choris-
mate synthase, and DHFR are the validated targets, but no data
yet indicate a particular protein as favored, and much interesting
work remains to be accomplished.
Acknowledgments
We thank M. Gottlieb for his insights and encouragement and
E. Holfels, V. Aitchison, H. McGuinness, and K. Zawisza for their
assistance in preparing this manuscript.
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