Download Isoprenoid biosynthesis in bacterial pathogens

Microbiology (2012), 158, 1389–1401
Review
DOI 10.1099/mic.0.051599-0
Isoprenoid biosynthesis in bacterial pathogens
Sinéad Heuston,1 Máire Begley,1 Cormac G. M. Gahan1,2,3 and Colin Hill1,2
Correspondence
1
Cormac G. M. Gahan
2
[email protected]
Department of Microbiology, University College Cork, Cork, Ireland
Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
3
School of Pharmacy, University College Cork, Cork, Ireland
Isoprenoid biosynthesis is essential for cell survival. Over 35 000 isoprenoid molecules have been
identified to date in the three domains of life (bacteria, archaea and eukaryotes), and these
molecules are involved in a wide variety of vital biological functions. Isoprenoids may be
synthesized via one of two independent nonhomologous pathways, the classical mevalonate
pathway or the alternative 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Given that
isoprenoids are indispensable, enzymes involved in their production have been investigated as
potential drug targets. It has also been observed that the MEP pathway intermediate 1-hydroxy-2methyl-2-(E)-butenyl 4-diphosphate (HMB-PP) can activate human Vc9/Vd2 T cells. Herein we
review isoprenoid biosynthesis in bacterial pathogens. The role of isoprenoid biosynthesis
pathways in host–pathogen interactions (virulence potential and immune stimulation) is examined.
Finally, the design of antimicrobial drugs that target isoprenoid biosynthesis pathways is
discussed.
Introduction
Isoprenoids are a large, diverse class of naturally occurring
organic chemicals which are essential for cell survival. Over
35 000 isoprenoid molecules have been identified to date in
the three domains of life (bacteria, archaea and eukaryotes), and they are involved in a wide variety of vital
biological functions. In bacteria, ubiquinone and menaquinone are involved in electron transport, while bactoprenols act as carbohydrate carriers in the biosynthesis of
peptidoglycan. Eukaryotic sterols such as cholesterol act as
membrane stabilizers and are precursors of bile acids.
Archaeal membrane lipids are composed of isoprenoid side
chains with ether links that join these side chains to the
glycerol backbone. In plants, the main isoprenoids are
those of the protective pigments such as carotenoids, which
make up part of the photosynthetic machinery. As well as
structural components isoprenoids also play a role in
hormone-based signalling, protein degradation and in the
regulation of transcription.
All isoprenoids are derived from the universal five-carbon
precursor isopentyl diphosphate (IPP) or its isomer dimethylallyl diphosphate (DMAPP), which may be synthesized via one of two independent pathways, the classical
mevalonate pathway or the alternative 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. The MEP pathway
intermediate 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMB-PP) has been shown to activate human Vc9/
Vd2 T cells. Expansion of this subset of T cells is observed
after infection with a broad range of microbial pathogens.
Herein we review isoprenoid biosynthesis in bacterial
051599 G 2012 SGM
pathogens. The role of the pathways in host–pathogen
interactions is examined and the design of drugs that target
isoprenoid biosynthesis is discussed.
Isoprenoid biosynthesis pathways
In animals, IPP biosynthesis proceeds exclusively via the
mevalonate pathway. The two isoprenoid biosynthesis
pathways are mutually exclusive in most bacterial species;
Listeria monocytogenes and certain Streptomyces species are
exceptions in that they have two complete pathways
(Boucher & Doolittle, 2000). Archaea utilize the mevalonate pathway (Matsumi et al., 2011).
The evolutionary origin of the two pathways remains controversial. A recent study by Lombard & Moreira (2011)
examined the origin and evolution of the enzymes of the
mevalonate pathway using phylogenomic analyses. Despite
the identification of several horizontal gene transfer events,
analyses support the hypothesis that the mevalonate pathway was ancestral in archaea, eukaryotes and bacteria. The
authors conclude that the mevalonate pathway is likely an
ancestral metabolic route in all three domains of life, and
was probably present in the last common ancestor of all
organisms.
The mevalonate pathway involves six enzymes which
convert acetyl-CoA and acetoacetyl-CoA into 3-hydroxy3-methylglutaryl-CoA (HMG-CoA), which is reduced to
mevalonate followed by phosphorylation to phosphomevalonate. A subsequent phosphorylation results in the
production of diphosphomevalonate, which is converted
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S. Heuston and others
into the C5 building blocks IPP or DMAPP. The MEP pathway requires eight enzymic steps, which proceed via the condensation of pyruvate and glyceraldehyde 3-phosphate to
produce 1-deoxy-D-xylulose 5-phosphate (DOXP). DOXP is
converted to MEP through the action of the enzyme DOXP
reductoisomerase (DXR) (IspC). 4-Diphosphocytidyl-2Cmethyl-D-erythritol (CDP-ME) is produced from the reaction of MEP with CTP, catalysed by YghP (IspD). The fourth
reaction, catalysed by YchB (IspE), produces 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate (CDP-ME2P). The
final reactions are catalysed by YghB (IspF), GcpE (IspG)
and LytB (IspH) to produce IPP (Fig. 1). The isomerase
enzyme which catalyses the interconversion of IPP and
DMAPP can be considered as common to both the MEP and
the mevalonate pathways.
The reader is referred elsewhere for in-depth reviews on the
elucidation and evolution of these pathways (Boucher &
Doolittle, 2000; Hunter, 2007; Lange et al., 2000; Matsumi
et al., 2011; Rohdich et al., 2001).
Isoprenoid biosynthesis in pathogens
Gram-negative bacteria
Escherichia coli. E. coli utilizes the MEP pathway for the
synthesis of IPP. Several groups have been involved in
deciphering the role and function of the individual MEP
enzymes, and this has led to significant insights into the
function of isoprenoid biosynthesis in E. coli and several
other bacteria (Altincicek et al., 2001; Eberl et al., 2002;
Herz et al., 2000; Rohdich et al., 1999, 2003a, b).
Mutational studies have proven the essentiality of yghP,
ychB, yghB, lytB and gcpE for growth (Altincicek et al.,
2001; Campos et al., 2001a; Campbell & Brown, 2002;
Freiberg et al., 2001; McAteer et al., 2001). It has been
shown that E. coli possesses a gene which encodes a type I
IPP isomerase (ipi), which maintains the supply of DMAPP
from IPP; however, this gene is not essential in E. coli
(Hunter, 2007). Genetic evidence for the branching of the
MEP pathway independently of IPI was obtained by
Rodrı́guez-Concepción et al. (2000), who showed that the
Fig. 1. Representation of the MEP and mevalonate pathways for IPP biosynthesis. MEP genes are shown with their historical
designation and current nomenclature (bold type). Statins inhibit HMGR, the rate-limiting enzyme of the mevalonate pathway.
The penultimate compound of the MEP pathway is HMB-PP, a non-peptidic antigen which is a potent activator of human Vc9/
Vd2 T cells. GA3P, glyceraldehyde 3-phosphate.
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Microbiology 158
Isoprenoid biosynthesis in bacterial pathogens
Table 1. Distribution of the MEP and mevalonate pathways amongst representative strains of Gram-positive and Gram-negative
pathogens
Pathogen
MEP
Mevalonate
Reference(s)
Gram-positive pathogens
Bacillus anthracis str. Sterne
Bacillus subtilis subsp. subtilis 168
Clostridium difficile 630
Clostridium botulinum B1 str. Okra
Clostridium perfringens E str. JGS1987
Enterococcus faecalis
L. monocytogenes EGDe
L. innocua Clip11262
Listeria seeligeri
Nocardia terpenica
Staph. aureus
Strep. pneumoniae
Strep. pyogenes
Gram-negative pathogens
B. abortus
Borrelia burgdorferi
Chlamydia trachomatis 434/Bu
Chlamydia pneumoniae
S. enterica serovar Typhimurium
E. coli O157 : H7
E. coli O127 : H6
F. tularensis
Legionella pneumophila
+
+D
+
+
+
2
+
2d
2d
+
2
2
2
2
2
2
2
2
+
+
+
+
2
+
+
+
*
Laupitz et al. (2004); Takagi et al. (2004)
*
*
*
Boucher & Doolittle (2000)
Begley et al. (2004)*
Begley et al. (2004)*
*
Shigemori et al. (1999)
Hammond & White (1970)
Wilding et al. (2000b)
Wilding et al. (2000b)
+D
2
+
+
+
+
+
+
2
2§
+
2
2
2
2§
2
2
+
P. aeruginosa
V. cholerae
K. pneumoniae subsp. pneumoniae MGH 78578
Bordetella pertussis Tahoma I
Haemophilus influenzae
H. pylori
Shigella flexneri
Shigella dysenteriae Sd197
Neisseria gonorrhoeae FA
Neisseria meningitidis
C. jejuni subsp. jejuni 81116
Y. enterocolitica
+
+
+
+
+
+
+
+
+
+
+
+
2
2||
2
2
2
2
2
2
2
2
2
2
Sangari et al. (2010)*
Boucher & Doolittle (2000)
Rohdich et al. (2001)
Eberl et al. (2003)
Cornish et al. (2006)*
*
*
Eberl et al. (2003)
Boucher & Doolittle (2000); Gophna et al.
(2006)
Putra et al. (1998)
Gophna et al. (2006)*
*
*
Boucher & Doolittle (2000)
Pérez-Gil et al. (2010)
*
*
*
Boucher & Doolittle (2000)
Gabrielsen et al. (2004)
*
*Sequences were analysed by performing BLASTP searches (using the web-based
Information website (http://www.ncbi.nlm.nih.gov/).
DPossess a DRL enzyme instead of DXR.
dPossess some of the pathway genes; missing the final two MEP genes.
§A type II IPP isomerase has been characterized.
||HMGR enzyme is present.
MEP pathway branched from the lytB gene. The study by
Puan et al. (2005) proposed that the flavodoxin I gene (fldA)
(a gene known to be essential in E. coli) has a role in the MEP
pathway by providing the reducing system required for the
enzymic activation of GcpE and LytB. Whilst much of the
genetic characterization has been performed using laboratory strains of E. coli, it is clear that pathogenic E. coli strains
(including enterohaemorrhagic, enteroaggregative, enterotoxigenic, enteroinvasive and enteropathogenic strains) also
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BLAST
program) on the National Centre for Biotechnology
possess the MEP pathway and lack genes encoding enzymes
of the mevalonate pathway (Table 1).
Brucella abortus. Analysis of the sequenced genome of B.
abortus identified homologues of seven of eight genes of the
MEP pathway. A homologue of the dxr gene, encoding
DXR, was not found. Sangari et al. (2010) identified a new
family of enzymes designated DXR-like (DRL) enzymes.
The DRL enzyme catalyses the same biochemical reaction
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S. Heuston and others
to produce MEP from DOXP, but has no overall homology to the DXR enzyme. A genomic library of B. abortus
was constructed, screened and used to complement a DXRdeficient E. coli mutant (again using the recombinant
strain constructed to utilize mevalonate). Cells capable of
mevalonate-independent growth were screened and further
characterized to confirm the existence of a DRL enzyme in B.
abortus.
Sangari et al. (2010) also delineated the phylogenetic
distribution of DRL and DXR. Upon examining the
distribution of complete MEP pathway enzymes with
respect to DRL sequences, three classes of bacteria were
determined: class A (B. abortus) possess DRL instead of
DXR sequences in their genomes, class B (L. monocytogenes,
Pseudomonas aeruginosa) possess sequences similar to both
DRL and DXR, and finally class C (archaea, Coxiella
burnetii) consists of bacteria with the DRL enzyme but
lacking MEP pathway enzymes.
Salmonella. The intracellular pathogen Salmonella enterica
serovar Typhimurium has the complete set of genes for the
MEP pathway of isoprenoid biosynthesis. Experiments by
Cornish et al. (2006) demonstrated that mutations in the
MEP pathway genes yghP, ychB, yghB, gcpE and lytB were
lethal.
Campylobacter jejuni. This Gram-negative foodborne
pathogen synthesizes IPP exclusively through the MEP
pathway. Gabrielsen et al. (2004) used an expression system
to characterize the YghP and YchB enzymes (referred to as
IspD and IspF, respectively). Analysis of the purified
proteins demonstrated the ability of YghB to convert MEP
to CDP-MEP, with YchB catalysing the conversion of CDPMEP to 2C-methyl-D-erythritol 2,4-cyclopyrophosphate
(MEcPP).
Vibrio cholerae. Analysis of the sequenced genomes
suggests that synthesis of IPP in this human intestinal
pathogen is via the MEP pathway; however, the gene for
the mevalonate pathway enzyme HMG-CoA reductase
(HMGR) is present in all four sequenced Vibrio species
(V. cholerae, Vibrio vulnificus, Vibrio parahaemolyticus
and Vibrio fischeri). Gophna et al. (2006) examined the
evolutionary history of hmgR within these species, and
their data suggest that hmgR was acquired by an ancestral
Vibrio in a single lateral gene transfer event. The function
of HMGR in Vibrio is as yet unknown, but evidence
strongly suggests that it is not involved in isoprenoid
biosynthesis, as the remaining genes of the mevalonate
pathway are not present. Furthermore, hmgR is located
on the minor chromosome, which is typically not
associated with metabolic functions, and transcription
has been shown to be extremely low under various
conditions tested (Gophna et al., 2006). Those authors
note that hmgR in Vibrio is under strong purifying
selection, which may hinder elucidation of the physiological role of HMGR.
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Helicobacter pylori. Genome analysis has revealed that H.
pylori utilizes the MEP pathway for IPP biosynthesis. Few
studies have functionally characterized the genes of the MEP
pathway in H. pylori, although recently Pérez-Gil et al.
(2010) expressed and characterized ispDF (corresponding to
yghPB) from this pathogen in an E. coli strain. The authors
demonstrated that this gene product is functional in E. coli
and may provide a target for the design of novel drug
moieties active against H. pylori.
Gram-positive bacteria
L. monocytogenes. Analysis of the L. monocytogenes
genome revealed that this pathogen possesses the full
complement of genes for both the MEP and the
mevalonate pathways (Begley et al., 2004). Initial analyses
of isoprenoid biosynthesis in L. monocytogenes were
performed with plasmid insertion mutants, two MEP
pathway mutants [gcpE (lmo1441) and lytB (lmo1451)] and
two mevalonate pathway mutants [hmgR (lmo0825) and
mvk (lmo0010)]. However, recent experiments in our
laboratory utilizing precise deletion mutants suggest that
HMGR may be essential for normal growth under laboratory conditions, despite the presence of an intact
MEP pathway (S. Heuston, unpublished data). We
previously created a double pathway mutant containing
deletions in the gcpE (lmo1441) and hmgR (lmo0825)
genes which cannot grow in the absence of exogenously
provided mevalonate, confirming the requirement of at
least one intact pathway for growth (Begley et al., 2008).
In silico analyses revealed that the closely related but nonpathogenic species Listeria innocua does not possess the
final two genes of the MEP pathway, which encode the
enzymes HMB-PP synthase (GcpE) and HMB-PP
reductase (LytB). Close analysis of the relevant genomic
regions indicates that L. innocua has lost these genes,
possibly during adaptation to a non-pathogenic life cycle.
This may suggest that the L. monocytogenes gcpE and lytB
genes are important for intracellular survival during
infection of the host but dispensable for a non-parasitic
life cycle (Begley et al., 2008).
Sangari et al. (2010) have reported that, in addition to a
functional Dxr, L. monocytogenes also possesses a drl gene.
They suggest that the presence of two enzymes with an
ability to catalyse the same reaction (DOXP into MEP)
could be advantageous in situations where isoprenoid
biosynthesis is absolutely required.
To date, HMGR is the only enzyme of the listerial isoprenoid
biosynthesis pathways that has been purified. Theivagt et al.
(2006) cloned hmgR in E. coli and demonstrated that the
purified enzyme was a class II HMGR exhibiting dual
enzyme specificity, i.e. an ability to utilize both NAD(H) and
NADP(H) in catalysis.
Staphylococcus aureus. Early studies to decipher the
route of IPP biosynthesis in Staph. aureus suggested that
it utilized the mevalonate pathway, as predicted by the
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Isoprenoid biosynthesis in bacterial pathogens
organism’s ability to incorporate radiolabelled mevalonate
into isoprenoids (Hammond & White, 1970). Bioinformatic analysis of the completed genomes now confirms the
presence of the mevalonate pathway genes. Wilding et al.
(2000a) verified the essential requirement of the mevalonate
pathway gene mvaA, which encodes the well-characterized
and rate-limiting enzyme HMG-CoA reductase (Bochar
et al., 1999). Initial attempts to delete this gene were
unsuccessful, and the mevalonate pathway mutant could
only be recovered when the medium was supplemented
with exogenous mevalonate (Wilding et al., 2000a).
Voynova et al. (2004) expressed the Staph. aureus Mvk
protein in E. coli and purified and characterized it. Parallel
experiments with heterologous eukaryotic mevalonate
kinases indicated that the Staph. aureus Mvk is much
less sensitive to feedback inhibition, although there are
similarities in structural features that are important for
catalytic function (Voynova et al., 2004). A study by
Balibar et al. (2009) investigated the cellular responses in
Staph. aureus to perturbations in the mevalonate pathway.
Three strains of Staph. aureus were constructed in which
mevalonate pathway genes (mvaS, mvaA and mvaK1/D/
K2) were placed under the control of an inducible
promoter. DNA microarray analysis was then used to
investigate the associated transcriptional changes. It was
observed that decreased expression of the mevalonate
pathway leads to downregulation of primary metabolism
genes, and upregulation of virulence factors and cell wall
biosynthetic genes. The authors suggest that this dramatic
shift in the transcriptome may be indicative of an altered
metabolic state, where growth is halted and cells are
primed for defence.
Other pathogens
Mycobacterium tuberculosis. Studies by Putra et al.
(1998) revealed that M. tuberculosis does not incorporate
radiolabelled mevalonate, suggesting the MEP pathway as
the primary source of IPP. This pathogen has been the
focus of numerous studies in recent years, and several of
the pathway genes have been characterized and shown to
play an essential role during in vitro growth, including dxs
(Bailey et al., 2002), dxr (ispC) (Brown & Parish, 2008),
yghP (ispD) (Eoh et al., 2009; Shi et al., 2007), ychB (ispE)
(Eoh et al., 2009) and yghB (ispF) (Buetow et al., 2007). An
extensive study by Brown et al. (2010) further demonstrated the essentiality of the dxs and gcpE (ispG) genes.
They also demonstrated the presence of two dxs genes
designated dxs1 and dxs2; dxs1 was shown to be essential
for in vitro growth. The authors hypothesize that HMB-PP
synthase is the rate-limiting step of the MEP pathway in M.
tuberculosis, as determined by measuring HMB-PP production to analyse the flux through the pathway. From this
study the evidence suggested HMB-PP synthase as the
bottleneck of the MEP pathway, with DOXP synthase controlling the flux through the pathway at the transcriptional
level (Brown et al., 2010).
Other pathogens. Analysis of the entire genome sequences
of Yersinia enterocolitica, Clostridium perfringens, Clostridium
botulinum, Klebsiella pneumoniae and Clostridium difficile
suggests that all of these pathogens utilize the MEP pathway,
with Enterococcus faecalis, Legionella pneumophila and Borrelia
burgdorferi shown to possess genes of the mevalonate pathway
for isoprenoid biosynthesis (Table 1).
Distribution of pathways
Bacillus subtilis, a non-pathogenic Gram-positive
model. Much of the functional analysis of the MEP
pathway in Gram-positive bacteria has been carried out in
Bacillus subtilis, and this work is likely to inform future
analyses of Gram-positive pathogens. Bacillus subtilis
possesses the full complement of MEP pathway genes.
Campbell & Brown (2002) have extensively characterized
the yacN gene, which is homologous to the E. coli yghB
(ispF) gene. Creation of a yacN deletion mutant could
only be achieved with conditional complementation
under the control of an inducible promoter. This gene
was shown to be important, as growth was severely
retarded in the absence of inducer. A deletion in this gene
resulted in phenotypic changes, with cells exhibiting
altered cell morphology. Bacillus subtilis was the organism
of choice in several studies to elucidate the structure and
function of the type II IPP isomerase which converts IPP
to DMAPP (Laupitz et al., 2004). Takagi et al. (2004)
identified the ypgA gene product as a type II isomerase,
which was found to be a non-essential gene in Bacillus
subtilis. Steinbacher et al. (2003) were the first to report
the crystal structure of type II isomerase from Bacillus
subtilis, paving the way for further studies to characterize
this as a novel antibiotic target.
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The distribution of the MEP and mevalonate pathways of
isoprenoid biosynthesis is highly complex, with a clear bias
towards the MEP pathway in bacteria and the classical
mevalonate pathway in eukaryotes and archaea (Lombard
& Moreira, 2011). Indeed, the MEP pathway is present in
pathogenic organisms as diverse as Listeria, Salmonella,
Mycobacterium, Brucella and Francisella, as highlighted in
Table 1, with isoprenoids synthesized by the mevalonate
pathway in Borrelia, Staphylococcus, Streptococcus, Lactobacillus casei and Lactobacillus plantarum (Jomaa et al.,
1999a). Interestingly, a metagenomic approach by Gill et al.
(2006) revealed that MEP pathway genes are abundant in the
indigenous microflora of the human distal gut microbiome,
perhaps reflecting the abundance of the MEP pathway
in bacteria in general. Furthermore, genome sequence
analyses have revealed that the common intestinal bacteria
Bacteroides, Bifidobacterium, Fusobacterium, E. coli and
Clostridium all possess genes of the MEP pathway. It is
currently unclear how or why the MEP pathway has evolved
in many bacterial species in preference to the ancestral
mevalonate pathway. Firstly, there is strong evidence to
suggest a role for the MEP pathway during the intracellular
survival of bacterial pathogens (Begley et al., 2008; Lai et al.,
2001; Shin et al., 2006; Eskra et al., 2001). Also, recent studies
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by Ershov (2007) analysed the energy efficiency of the MEP
pathway in comparison with the mevalonate pathway. A
distinct difference in the ratio of IPP to DMAPP was
observed, with the MEP pathway synthesizing a 5 : 1 ratio of
IPP to DMAPP and the mevalonate pathway generating a
3 : 7 ratio of IPP to DMAPP, highlighting the reduced energy
consumption of the MEP pathway. Anaerobic bacteria such
as bifidobacteria seem to preferentially utilize this pathway,
and it may be that the energetics of this pathway are
favoured under the anaerobic conditions in the gut.
Role of isoprenoid pathways in host–pathogen
interactions
Virulence potential
Experiments using the mouse model of infection demonstrated that L. monocytogenes gcpE and lytB mutants were
impaired in virulence potential following intraperitoneal
infection (Begley et al., 2004, 2008). A double pathway
mutant (DgcpEDhmgR) was not recovered 3 days postinfection, indicating that the intracellular levels of mevalonate were not sufficient to sustain growth of a strain
deficient in both IPP biosynthesis pathways (Begley et al.,
2008). An independent study by Schauer et al. (2010)
reported a requirement for the MEP pathway ychB
(lmo0190) during in vivo survival of L. monocytogenes.
When BALB/c mice were inoculated intravenously, a ychB
mutant demonstrated impaired virulence potential in the
liver and spleens compared with the wild-type. The findings
of both of these studies suggest a requirement for the L.
monocytogenes MEP pathway during host infection.
The MEP pathway has also been implicated in the virulence
of other pathogens. A gcpE transposon mutant of M.
tuberculosis exhibited decreased colonization ability in the
liver and intestine of BALB/c mice following intraperitoneal infection (Shin et al., 2006). A study by Lai et al.
(2001) demonstrated that the K. pneumoniae dxr gene was
expressed following intraperitoneal infection of BALB/c
mice. Expression studies using a GFP reporter system
revealed that the B. abortus dxs gene was activated during
survival within RAW macrophages. The authors of that
study proposed that the expression of this gene leads to the
accumulation of antioxidants (produced via the MEP
pathway), which allows for initial survival in the oxidative
environment within the phagosome of the macrophage
(Eskra et al., 2001).
It has been shown that host cholesterol is important for cell
entry and intracellular proliferation of Salmonella. Garner
et al. (2002) demonstrated an accumulation of host cholesterol into the Salmonella-containing vacuoles (SCVs)
within murine macrophages and epithelial cell lines. Further studies by Catron et al. (2004) investigated the effect of
inhibiting the host mevalonate pathway. This involved the
targeted inhibition of the host HMG-CoA reductase
(HMGR), the rate-limiting step of the mevalonate pathway. Statins (cholesterol lowering drugs) were used to
inhibit the host mevalonate pathway. Inhibition of the host
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mevalonate pathway resulted in reduced intracellular growth
by the bacteria in a mouse model and infected macrophage
cell line. These results demonstrate the requirement of
Salmonella typhimurium for the host mevalonate pathway
during intracellular growth.
Evidence suggests that the MEP pathway may be involved
in combating oxidative stress. Interestingly Ostrovsky et al.
(1998) found that 2C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC), an intermediate of the MEP pathway,
accumulated in mycobacteria and corynebacteria in response to oxidative stress conditions. Carotenoids have been
shown to elicit a protective effect against reactive oxygen
species (Ostrovsky et al., 1998). A study of Chlamydia
trachomatis demonstrated the requirement for 2C-methylD-erythritol-2,4-cyclopyrophosphate (MEC), a molecule
which instigates intracellular development through disruption of histone-like protein Hc-1–DNA interactions
(Grieshaber et al., 2004). M. tuberculosis MEP pathway
mutants (DlytB and Ddxs) were impaired in their ability to
survive within the phagosome, and therefore this pathway
has been linked with the intracellular stress response in this
bacterium (Pethe et al., 2004). These studies demonstrate
that isoprenoids or derivatives of isoprenoids allow adaptation to the host environment through enhanced stress
resistance or other mechanisms. It is worth noting that the
DOXP of the MEP pathway not only is involved in IPP
biosynthesis but also is an intermediate for thiamine
(vitamin B1) and pyridoxol (vitamin B6) biosynthesis, both
of which are essential for human health (Hill et al., 1996).
Immune stimulation
In humans and primates, Vc9/Vd2 T cells constitute
0.5–5 % of peripheral blood T cells (Caccamo et al.,
2006). The expansion of this subset of T cells has been well
documented in the blood plasma of patients suffering from
a wide range of microbial infections, including listeriosis,
salmonellosis, malaria and tuberculosis (Morita et al.,
2007). Early studies implicated IPP biosynthesis in the
reactivity of Vc9/Vd2 T cells, but only bacteria utilizing
the MEP pathway could elicit this immune response
(Jomaa et al., 1999a). The stimulatory capacity of several
MEP pathway mutants in E. coli was analysed and a significant difference was observed between a DgcpE mutant
which exhibited a reduced capacity to elicit an immune
response in contrast to the highly immunostimulatory
DlytB mutant. Analysis of DlytB mutant extracts identified
HMB-PP as the most potent stimulator of Vc9/Vd2 T cells,
with a 10 000-fold increase in the stimulatory capacity of
this compound compared with IPP (Altincicek et al., 2001;
Eberl et al., 2002; Hecht et al., 2001; Hintz et al., 2001;
Reichenberg et al., 2003). These findings made it clear that
the inability of the pathogenic bacteria Staphlococcus,
Streptococcus and Borrelia to stimulate Vc9/Vd2 T cells
was due to the lack of an MEP pathway and the absence of
HMB-PP (Jomaa et al., 1999a). The ability of isoprenoid
biosynthetic pathways to produce non-peptidic compounds
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Isoprenoid biosynthesis in bacterial pathogens
and stimulate Vc9/Vd2 T cells has been reviewed elsewhere
(Eberl et al., 2003; Bonneville & Scotet, 2006).
L. monoctyogenes is an unusual pathogen in possessing two
complete pathways for IPP biosynthesis. cd T cell bioactivity from bacterial extracts was calibrated against a
standard of synthetic HMB-PP and the resultant bioactivity
was expressed as equivalents of HMB-PP. A DgcpE mutant
did not produce HMB-PP, resulting in a decreased
stimulatory capacity below wild-type levels, but a DlytB
mutant (HMB-PP+) exhibited increased capacity to
stimulate an immune response as compared with the
wild-type (Begley et al., 2004). Similarly, a study by Brown
et al. (2010) also demonstrated the increased accumulation
of HMB-PP in an M. tuberculosis strain overexpressing
gcpE. Begley et al. (2008) investigated the possibility of
restoring the MEP pathway in L. innocua. The missing final
two genes of the MEP pathway were restored by introducing gcpE on its own and gcpE together with the lytB
gene. The presence of gcpE alone conferred the capacity to
produce HMB-PP and thereby stimulate Vc9/Vd2 T cells, a
phenotype that does not exist for the wild-type L. innocua
strain (Begley et al., 2008). The precise mechanism
underpinning the stimulation of Vc9/Vd2 T cells by
HMB-PP is unclear. The elucidation of this process is
complicated by the fact that this particular subset of T cells
is only present in humans and higher primates.
Several hypotheses on the role of Vc9/Vd2 T cells in the
immune response have been proposed, including killing of
infected target cells (Bonneville & Scotet, 2006), cytokine
production, maturation of dendritic cells, and an ability to
act like antigen-presenting cells (Bonneville et al., 2010;
Eberl et al., 2009). Initial studies by Bonneville & Scotet
(2006) attempted to determine the mechanisms by which
HMB-PP stimulates Vc9/Vd2 T cells. This study demonstrated the cytotoxic effects of Vc9/Vd2 cells in the killing
of Mycobacterium bovis BCG-infected dendritic cells but
did not determine the mode of HMB-PP presentation to
the Vc9/Vd2 T cells. Eberl and colleagues then confirmed
the functioning of monocytes as ‘feeder’ cells in the
stimulation of Vc9/Vd2 T cells with HMB-PP during
bacterial infections (Eberl et al., 2009). A recent study by
Davey et al. (2011) delved further into the relationship
between Vc9/Vd2 T cells and HMB-PP within the host.
They demonstrated that HMB-PP is in fact released from
infected neutrophils into the microenvironment. This
study was performed using the non-pathogenic L. innocua,
which cannot produce HMB-PP, a strain with the ability to
produce HMB-PP conferred by the presence of the gcpE
gene, and a strain of Mycobacterium smegmatis engineered
to overproduce HMB-PP. They demonstrated that the
capacity of Vc9/Vd2 T cells to respond to infected neutrophils is dependent on cell to cell contact with monocytes
coupled with the ability of the invading bacteria to produce
HMB-PP, demonstrating that only HMB-PP-producing
bacteria could activate the surrounding Vc9/Vd2 T cell
population (Fig. 2). The cell to cell contact between monocytes and Vc9/Vd2 T cells proved crucial in eliciting an
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Fig. 2. Stimulation of an immune response by HMB-PP. The host
synthesizes isoprenoids via the mevalonate pathway. An intracellular pathogen engulfed within a host phagocytic cell utilizes the
MEP pathway for essential isoprenoid biosynthesis. Isoprenoid
biosynthesis via the MEP results in the production of HMB-PP.
Following phagocytosis of the gut pathogen, HMB-PP is released
into the microenvironment. HMB-PP is then recognized by the
Vc9/Vd2 T cells (manner of recognition remains undetermined) and
these cells become stimulated in response to HMB-PP. The
stimulatory response has been found to be optimal in the presence
of direct cell to cell contact between Vc9/Vd2 T cells and
monocytes. This figure was adapted from Davey et al. (2011).
optimum response to the phagocytosed bacteria from the
infected neutrophil (Davey et al., 2011). However, the exact
mechanism by which HMB-PP is recognized by the Vc9/
Vd2 T cell still remains to be determined. Interestingly,
recent evidence has shown that aminobisphosphonate and
alkylamine (isoprenoid biosynthesis) inhibitors can indirectly stimulate Vc9/Vd2 T cells. This highlights that there
are multiple ways to stimulate Vc9/Vd2 T cells, and
emphasizes the requirement to characterize both direct and
indirect pathways for Vc9/Vd2 T cell stimulation (Wang
et al., 2011a).
Isoprenoid biosynthesis pathways as drug targets
Mevalonate pathway drug targets
It is significant that human/mammalian isoprenoid biosynthesis is the target of several FDA-approved drugs,
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S. Heuston and others
including those used in the treatment of cancer (taxol),
bone disease (bisphosphonates) and hypercholesterolaemia
(statins) (Buhaescu & Izzedine, 2007). The potential of the
mevalonate pathway as a drug target has been reviewed in
detail by Buhaescu & Izzedine (2007). In humans, cholesterol biosynthesis proceeds via the mevalonate pathway,
which is responsible for the production of the IPP precursor molecule. Some of the best-characterized and most
effective inhibitors of IPP biosynthesis are the cholesterollowering statins. Statins disrupt a reaction in the mevalonate pathway by binding to and inhibiting HMGR, the
rate-limiting enzyme for cholesterol biosynthesis in mammals. Nitrogenous bisphosphonates (NBPs) such as pamidronate and zoledronate, which are predominantly used to
treat bone disease, also target the mevalonate pathway by
inhibiting farnesyl pyrophosphate synthase. The IPP isomerases have been considered as potential targets; however,
as humans possess the type I isomerase, specific inhibitors of
the type II enzyme could function to target pathogenic bacteria such as multidrug-resistant Staphylococcus (Kuzuyama
et al., 2000).
The MEP pathway as a drug target
Enzymes of the MEP pathway have been classified as
metabolic chokepoints, meaning that they can consume or
generate a specific product and their function cannot be
compensated for by any other enzyme (Hasan et al., 2006).
This, together with the fact that IPP synthesis in humans
proceeds via the mevalonate pathway (so there are no
human MEP enzyme homologues), has resulted in the
MEP pathway of pathogens being considered as a drug
target (reviewed by Ershov, 2007; Obiol-Pardo et al., 2011;
Rohdich et al., 2005; Testa & Brown, 2003).
The identification and characterization of E. coli DXR
catalytic residues was described by Kuzuyama et al. (2000),
and further characterization studies have determined the
crystal structure of E. coli DXR when complexed with
cofactors (NADPH) and fosmidomycin (Steinbacher et al.,
2003). Solving the crystal structures of these enzymes was a
crucial step to pave the way for drug design. In 1999, Jomaa
and colleagues provided evidence that the MEP pathway is
used by the malaria parasite Plasmodium falciparum, and
that fosmidomycin and its analogue FR-900098 had the
ability to inhibit DXR from Plasmodium falciparum in
a dose-dependent manner. Both inhibitors cured mice
infected with the rodent malaria parasite Plasmodium
vinckei, demonstrating their use as anti-malarial compounds (Jomaa et al., 1999b). Clinical trials proved that
fosmidomycin was effective in the treatment of patients
suffering from uncomplicated Plasmodium falciparum
malaria (Lell et al., 2003). The anti-malarial activity of
FR-900098 was approximately twofold greater than that of
fosmidomycin. Fosmidomycin treatment led to a fast
reduction in fever and clearance of the parasite, but was
precluded as a monotherapy due to inefficient elimination
of the parasite. A combination of fosmidomycin with
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clindamycin could result in more synergistic effects (Singh
et al., 2007). Kurz et al. (2006) reported the synthesis of
chain-substituted pivaloyloxymethyl ester derivatives of
fosmidomycin and FR900098 with comparable in vitro
antimalarial activity. Kuntz et al. (2005) synthesized two
phosphonohydroxamic acids which showed inhibitory
activity towards DXR as well as antibacterial activity
against E. coli in vitro. Indeed, Behrendt et al. (2011) very
recently reported the antiplasmodial activity of IspC/Dxr
inhibitors.
YchP, YchB and YghB have all been shown to be essential
in E. coli, Bacillus subtilis, Haemophilus influenzae and
M. tuberculosis (Buetow et al., 2007; Campbell & Brown,
2002; Campos et al., 2001a, b; Freiberg et al., 2001). Several characterization and screening studies have analysed
these MEP pathway enzymes as potential drug targets.
Illarionova et al. (2006) described the enzyme-assisted
preparation of isoprenoid biosynthesis intermediates and
high-throughput methods for screening of IspC/Dxr, IspD/
YghP and IspE/YchB against large compound libraries. 13C
NMR assays were subsequently employed to verify hits via
direct detection of the primary reaction product. The IspD/
YghP enzyme of M. tuberculosis was cloned and expressed
in an E. coli system, and the purified recombinant protein
was subjected to biochemical and enzymic characterization
in order to gain insight into the structure of this protein
for therapeutic drug design (Shi et al., 2007). A recent
publication by Tsang et al. (2011) details the kinetic characterization of the IspD enzyme of Francisella tularensis,
and their results validate the targeting of this enzyme for
novel therapeutics against F. tularensis. Detailed biochemical characterization and crystal structure models (to
analyse substrate-binding sites) for YchB were determined
using an efficient expression system with YchB from the
thermophilic bacterium Aquifex aeolicus. Sequence–structure comparisons revealed that the active site and ligand
interactions are highly conserved for YchB and that A.
aeolicus YchB represents a suitable model for the human
pathogen M. tuberculosis (Sgraja et al., 2008). A. aeolicus
IspE has proved invaluable for providing structural information in the structure-based design approach for the
generation of IspE inhibitors for organisms such as Plasmodium falciparum and M. tuberculosis (Crane et al., 2008;
Hirsch et al., 2008). Co-crystal structure analysis (Crane
et al., 2008; Hirsch et al., 2008) and structure–activity
relationships (SARs) (Hirsch et al., 2008) of A. aeolicus
IspE have validated the binding mode of several new
inhibitors.
Buetow et al. (2007) determined the crystal structure of M.
smegmatis YghB, which has a high degree of similarity to
YghB of M. tuberculosis. This study allowed the identification of active sites and binding pockets, and provides a
template for structure-based inhibitor design (Buetow et al., 2007).
Ramsden et al. (2009) utilized the structure of E. coli IspF
bound to CDP to identify features considered important
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Isoprenoid biosynthesis in bacterial pathogens
for ligand binding and affinity. The rigid nucleotide–Zn2+
binding pocket of the active site was noted. Subsequently, a
computational docking approach was employed to screen,
in silico, a database of approximately one million commercially available compounds against the nucleotide–
Zn2+ binding pocket. Selected hits from the virtual screen
were examined experimentally with X-ray crystallography
and a surface plasmon resonance assay, and two weakly
binding ligands were identified (Ramsden et al., 2009).
These initial results prompted additional experiments
using a more focused set of compounds to probe the
IspF cytidine and Zn2+ binding sites, and yielded a ligand
with low micromolar binding affinity. An additional study
by Jawaid et al. (2009) examined the IspF enzyme of F.
tularensis. The enzyme was cloned and expressed in E. coli,
the recombinant protein was then purified and enzymically
characterized, and subsequent analysis showed that IspF is
an excellent target for antibiotic development.
The 3D structures for YghP, YchB and YghB are available
for M. tuberculosis, Plasmodium Falciparum and C. jejuni,
respectively (Miallau et al., 2003; Richard et al., 2001;
Steinbacher et al., 2003). The crystal structures for these
enzymes in E. coli have also been solved; however, despite
these developments, no successful inhibitors for these three
enzymes have been identified to date (Miallau et al., 2003;
Richard et al., 2001; Steinbacher et al., 2003).
The last two enzymes of the MEP pathway, IspG/GcpE and
IspH/LytB, are 4Fe–4S proteins, which are involved in
essential reduction steps in isoprenoid biosynthesis. The
crystal structure for IspH is known for both E. coli
(Gräwert et al., 2010) and A. aeolicus (Rekittke et al., 2008).
Pyridine-based inhibitors of IspH have undergone spectroscopic and chemical investigation to elucidate their
inhibitor functions (Wang et al., 2011b). Indeed, Wang
et al. (2010) screened a series of small molecules using EPR
spectroscopy and put forward the proposal that diphosphonates inhibit IspH through interaction with the active
site, thereby preventing HMB-PP from binding to the 4Fe–
4S cluster.
There are two classes of IPP isomerases: humans and
animals possess the type I isomerase, and the majority of
pathogenic bacteria possess the type II enzyme. So far, no
specific type II inhibitors have been identified, but this
enzyme has been pinpointed as an attractive target in
infections by Gram-positive cocci, especially multidrugresistant Staph. aureus (Rohdich et al., 2005).
Both farnesyl diphosphate synthase (FPPS) and undecaprenyl diphosphate synthase (UPPS) enzymes are essential
for bacterial cell growth. FPPS catalyses the condensation
of DMAPP with IPP to form geranyl diphosphate (GPP),
and UPPS elongates FPP. UPPS is of particular interest, as
like the MEP pathway enzymes it is also absent in humans.
Interestingly, Durrant et al. (2011) described a novel
screening approach to identify non-bisphosphonate FPP
synthase inhibitors which also showed inhibitory properties against UPP synthase enzymes. These compounds
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were shown to be effective at micromolar concentrations
(Durrant et al., 2011), and furthermore they appear to be
more amenable to clinical use than their bisphosphonate
counterparts, as they are less vulnerable to rapid removal
from the circulatory system (Jahnke et al., 2010).
It is estimated that approximately 70 % of the human gut
microbiome is unculturable under standard laboratory
conditions, but with recent and rapid advances in cultureindependent sequencing techniques the genetic makeup of
previously uncharacterized gut commensals has now been
determined. Metagenomic analysis suggests that commensals utilize the MEP pathway (Gill et al., 2006; S. Heuston,
unpublished data). Although the MEP pathway represents
an attractive and valid target for pathogenic bacteria,
careful consideration is required, as drugs targeting this
pathway will also likely affect the human gut commensal
population, possibly resulting in an undesirable effect on
the human superorganism.
Conclusions and future directions
The MEP pathway is the principal means of IPP biosynthesis
in the majority of human pathogens. The penultimate
intermediate of the MEP pathway (HMB-PP) is known to be
the most potent activator of human Vc9/Vd2 T cells. Several
elegant studies have demonstrated the ability of MEPutilizing bacteria to stimulate Vc9/Vd2 T cells. The in vitro
and in vivo experimental analysis discussed in this review
indicates a link between the MEP pathway and the intracellular survival of pathogenic bacteria.
IPP biosynthesis in humans proceeds via the mevalonate
pathway, and so the development of novel drugs that target
the MEP pathway would result in a new class of broadspectrum antibiotics. However, as discussed in this review,
extensive metagenomic analysis of the human gut microbiome has demonstrated an over-representation of the
MEP pathway in gut bacteria. As a result, drugs targeting
the MEP pathway would consequently target gut bacteria
and possibly result in detrimental effects on the human host.
The targeting of MEP pathway enzymes may need to be
re-evaluated from the perspective of the host, and due
consideration should be given to dual therapies which would
involve MEP-targeted antibiotics used in conjunction with
probiotic therapies (to restore the host microbiome).
One unanswered question is why gut bacteria favour the
MEP pathway. The MEP pathway could be favoured due to
its energy requirements. Ershov (2007) discussed the
energy efficiency of the MEP pathway in comparison with
the mevalonate pathway. A distinct difference in the ratio
of IPP to DMAPP was noted, with the MEP pathway
synthesizing a 5 : 1 ratio of IPP to DMAPP and the
mevalonate pathway generating a 3 : 7 ratio. These factors
could have important implications in the evolutionary
selection of the MEP pathway. Anaerobic bacteria such as
bifidobacteria seem to preferentially utilize this pathway
(Ershov, 2007).
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S. Heuston and others
Evidence suggests a role for the MEP pathway during the
intracellular survival of bacterial pathogens. Interestingly,
several extracellular pathogens appear to utilize the mevalonate pathway rather than the MEP pathway (Staph.
aureus, Streptococcus pneumoniae and Streptococcus pyogenes). Studies in both Staph. aureus and Strep. pyogenes
have demonstrated their inability to activate Vc9/Vd2 T
cells (Jomaa et al., 1999a). Furthermore, an adaptation to a
non-pathogenic extracellular life cycle in L. innocua has
clearly led to the loss of two genes of the MEP pathway, with
L. innocua possessing only the mevalonate pathway (Begley
et al., 2008). MEP-utilizing bacteria can elicit an immune
response through the production of the non-peptidic
penultimate compound of the MEP pathway, HMB-PP,
but it is not clear whether this immuno-stimulatory capacity
is advantageous to the bacterium residing in the host. As
the MEP pathway is over-represented in the human gut
metagenome, this immune stimulation is not limited to gut
pathogens but extends to autochthonous gut microbes. This
suggests that the stimulatory capacity of the bacteria may in
fact induce a state of tolerance within the hosts and enable
the bacteria to evade the immune system. This particular
subset of T cells can function as a bridge linking innate and
adaptive immune responses. However, the exact reason why
bacteria would produce HMB-PP to stimulate an immune
response still remains unclear.
Future studies may be best focused on determining a role
for the MEP pathway during the gastrointestinal phase of
the bacterial life cycle. The ability of the MEP pathway to
stimulate an immune response from the perspective of the
host warrants further characterization, but these studies
cannot be performed in murine models, as mice do not
possess the Vc9/Vd2 subset of T cells. Future studies would
need to be performed in primate models such as Rhesus
Macaques (Macaca mulatta) and Night Monkeys (Aotus
nancymaae). Recent studies have contributed greatly to
elucidating the methods by which HMB-PP is released
from infected cells, but the exact mechanisms by which
HMB-PP is recognized by Vc9/Vd2 T cells still remain
unclear.
To our knowledge there are very few studies which
document the levels of IPP production in bacterial mutant
strains, with most experimental data correlating HMB-PP
concentration with the bioactivity of Vc9/Vd2. Effective
methods to measure the production of pathway intermediates as well as end products would greatly increase our
understanding of the functioning of these pathways. A
fundamental understanding of the flux or regulation of
these pathways during different stages in the bacterial life
cycle will also be essential. Studies to determine whether
some of the dominant intracellular gut pathogens have the
ability to utilize the host mevalonate pathway during
intracellular survival would be very useful to help
determine the roles of these pathways in vivo.
In conclusion, numerous elegant studies, largely biochemical and immunological, have led to significant advances in
1398
our understanding of isoprenoid biosynthesis pathways.
Many questions remain unanswered regarding the use of
these pathways by human pathogens and host commensals,
and it is highly likely that isoprenoids have as-yet-unidentified
and possibly unexpected roles in bacterial physiology.
Acknowledgements
We would like to acknowledge the funding received from the Irish
Government under the National Development Plan 2000–2006 and
the funding of the Alimentary Pharmabiotic Centre by the Science
Foundation of Ireland Centres for Science Engineering and
Technology (CSET) programme. S. H. was the recipient of an
EMBARK postgraduate research scholarship from the Irish Research
Council for Science Engineering and Technology (IRCSET).
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