Introduction
Trypanosoma brucei is a single-celled protozoan pathogen that causes human and animal
trypanosomiasis and incurs devastating health and economic burdens in Africa. Together
with the related parasites T. cruzi and Leishmania spp., which cause Chagas disease
and leishmaniasis, respectively, over 8 million people are affected annually worldwide
[1]. These parasites alternate between a mammalian host and the insect vector and
undergo extensive developmental changes during their life cycle, including changes
in surface coat, gene expression, metabolism, and organelle morphology and function.
They also have elaborate mechanisms of gene regulation that control the expression
of genes involved in host immune evasion during infection. The control of developmental
changes and immune evasion mechanisms entails a complex network of signaling and regulatory
processes that includes phosphatidylinositol (PI) phosphates (PIP, also called phosphoinositides)
and inositol phosphates (IP) [2–8]. PIPs and IPs are ubiquitous in eukaryotes and
consist of a subset of molecules containing mono or poly phosphorylated inositol (Fig
1A). Whilst PIPs are a class of phospholipids generally associated with cellular or
organellar membranes and produced via phosphorylation of PI, IPs are soluble molecules
produced as a result of PIP hydrolysis by phospholipase enzymes. PIPs and IPs interact
with proteins or RNA and regulate numerous cellular functions in eukaryotes. As detailed
below, these metabolites and related enzymes function as a regulatory system with
essential roles in T. brucei metabolism and development [6], trafficking and organelle
biogenesis [9–11], Ca2+ signaling [12], and immune evasion mechanisms [5, 7].
10.1371/journal.ppat.1008167.g001
Fig 1
PIP and IP synthesis and regulation in T. brucei.
(A) Structure of PIP2 indicated by the inositol ring (black hexagon), phosphates (red
circles), and DAG with fatty acid chain. PLC cleaves PIP2 and produces diacylglycerol
and IP3. Black arrows indicate phosphate and inositol. The yellow arrow indicates
the site of PLC cleavage, which occurs between DAG and phosphate sn1. The green arrow
indicates the directionality of the PLC reaction. (B) The number of genes involved
in PIP and IP synthesis, signaling (includes PLC and IP3 receptors), and PIP and IP
kinases and phosphatases in eukaryotes and prokaryotes. The size of the black circles
indicates the number of genes in each category. (C) Synthesis of PIPs and IPs based
on T. brucei predicted and characterized enzymes. Enzymes, whose regulatory functions
are discussed here, are indicated in blue. PIP-Pase indicates enzymes that dephosphorylate
PIPs at positions 3, 4, or 5 of the inositol ring. It includes PIP5Pase, whose catalytic
activity is detailed below in D. Metabolite short names are used for simplicity. (D)
Regulation of VSG silencing by PIP5Pase. PIP5Pase dephosphorylates the 5-phosphate
(green circle) of PIP3 and prevents this metabolite binding to RAP1, which preserves
RAP1 function (and likely other proteins) in ES chromatin organization. Catalytic
inactivation of PIP5Pase results in PIP3 binding to RAP1, which affects ES chromatin
organization and results in transcription of VSG genes. 1, diacylglycerol kinase;
2, cytidine diphosphate-diacylglycerol synthase; 3, phosphatidylinositol synthase;
70 bp, 70 base pair repeats; Ath, Arabidopsis thaliana; DAG, diacylglycerol; ER, endoplasmic
reticulum; ES, expression site; ESAG, expression site associated genes; Hsp, Homo
sapiens; I, myo-inositol; IMPase, inositol monophosphatase; IP, inositol phosphate;
IP1, D-myo-inositol 1-monophosphate; IP2, D-myo-inositol 1,4-diphosphate; IP3, D-myo-inositol
1,4,5-triphosphate; IP4, D-myo-inositol 1,3,4,5-tetrakisphosphate; IP5, D-myo-inositol
1,2,3,4,5-pentakisphosphate; IP5Pase, inositol polyphosphate 5-phosphatase; IP6, D-myo-inositol
1,2,3,4,5,6-hexakisphosphate; IP6K, inositol hexakisphosphate kinase; IP7, D-myo-inositol
5-diphospho 1,2,3,4,6-pentakisphosphate; IPMK, inositol polyphosphate multikinase;
Mtb, Mycobacterium tuberculosis; PIP, phosphatidylinositol phosphate; PIP1, phosphatidylinositol
4-phosphate; PIP2, phosphatidylinositol 4,5-biphosphate; PIP3, phosphatidylinositol
3,4,5-triphosphate; PIP5K, phosphatidylinositol phosphate 5-kinase; PIP5Pase, phosphatidylinositol
phosphate 5-phosphatase; PIP-Pase, phosphatidylinositol phosphate phosphatases; PLC,
Phospholipase C; PM, plasma membrane; Pol I, RNA polymerase I; PP-IP4, D-myo-inositol
5-diphospho 1,3,4,6-tetrakisphosphate; RAP1, repressor-activator protein 1; Sce, Saccharomyces
cerevisiae; sn1, unimolecular nucleophilic substitution; Tbr, T. brucei; Ttm, Thermus
thermophilus; VSG, variant surface glycoprotein.
From structural molecules to regulators
The T. brucei genome encodes four enzymes involved in the synthesis of inositol and
PI, one inositol symporter, 23 PIP or IP kinases and phosphatases, one phospholipase
C (PLC), and one inositol trisphosphate (IP3)/ryanodine receptor (IP3RyR) [13] (Fig
1B). T. brucei synthesizes PI in the endoplasmic reticulum (ER) and Golgi [14, 15],
which is then distributed to other subcellular compartments by mechanisms yet unknown.
At the plasma membrane inner leaflet, PLC cleaves phosphatidylinositol 4,5-bisphosphate
(PIP2) and generates diacylglycerol and IP3 (Fig 1A and 1C), and the latter is further
phosphorylated or dephosphorylated by IP kinases and phosphatases, respectively [6,
16, 17] (Fig 1C). This set of synthesis, cleavage, and modifying enzymes (hereafter
referred as PIP/IP-related proteins) produces at least 11 different PIP and IP metabolites
(Fig 1C), some of which have been detected in T. brucei via immunofluorescence or
mass spectrometry methods [7, 14, 15] or predicted to exist based on in vitro enzymatic
studies [6, 13, 18]. T. brucei PIP and IP kinases and phosphatases with different
specificities are distributed in distinct subcellular locations, e.g., plasma membrane,
endosomes, and nucleus [5, 7, 9, 10, 12] (Table 1). The subcellular distribution of
PIPs, IPs, and related proteins in T. brucei indicates that they function as a regulatory
system in addition to their role in the synthesis of membrane or glycoconjugate structures.
This is evidenced by the numerous cellular processes that are affected by knockdown
or mutation of genes encoding PIP/IP-related proteins [6, 9, 10, 12, 18, 19] (Table
1). This regulatory system relies primarily on the activity of PIP and IP kinases
and phosphatases, which control the phosphorylation and turnover of PIP and IP metabolites,
and on the ability of these metabolites to interact with proteins and thus regulate
protein function. Similar to T. brucei, other single-celled eukaryotes, such as Plasmodium
sp., Giardia sp., and Saccharomyces sp. (baker’s yeast), seem to have a PIP/IP regulatory
system (Fig 1B). Notably, this regulatory system seems to be absent in prokaryotes,
which only have enzymes involved in the synthesis of inositol and PI, which are typically
incorporated into glycoconjugates [20]. On the other hand, metazoans have a PIP/IP
regulatory system with expanded and diversified gene content encoding PIP and IP kinases,
phosphatases, and phospholipases, which likely reflects the diversity of cell types,
developmental processes, signaling, and regulation in metazoans that are absent in
unicellular eukaryotes [21]. This scenario indicates that PIPs, IPs, and related proteins
function as a regulatory system that diversified with the complexity of eukaryotic
organisms.
10.1371/journal.ppat.1008167.t001
Table 1
Regulatory roles of PIP and IP enzymes in T. brucei.
Gene ID
Enzyme
Regulatory process
Localization
Reference
Tb927.4.1620
PIP5K
VSG gene exclusive expression and switching
Plasma membrane and endosomes
[7]
Tb927.11.6270
PIP5Pase
VSG gene exclusive expression
Nucleus
[5, 7]
Tb927.11.5970
PLC
VSG gene exclusive expression
Plasma membrane
[7]
Tb927.9.12470
IPMK
Metabolic switch from glycolysis to oxphos and development of BFs to PFs
Plasma membrane and cytosol
[6]
Tb927.8.2770
IP3RyR
Intracellular Ca2+ from acidocalcisomes
Acidocalcisomes
[12]
Tb927.4.1140
PI4K
Protein trafficking and Golgi maintenance and structure
Golgi complex
[11]
Tb927.8.6210
PI3K
Golgi complex segregation
Golgi complex
[10]
Tb927.11.1460
PI3P5K
Protein trafficking and multivesicular body degradation
Endosome and lysosomes
[9]
Gene IDs are from TriTrypDB. IP3RyR, inositol trisphosphate/ryanodine receptor; IPMK,
inositol polyphosphate multikinase; PI3K, phosphatidylinositol 3-kinase; PI3P5K, phosphatidylinositol
3-phosphate 5-kinase; PI4K, phosphatidylinositol 4-kinase; PIP5K, phosphatidylinositol
phosphate 5-kinase; PIP5Pase, phosphatidylinositol phosphate 5-phosphatase; PLC, phospholipase
C
IP signaling and regulation: Beyond second messenger function
In metazoans, IP3 functions as a second messenger, which controls Ca2+ release from
the ER via an IP3RyR and activates downstream signaling cascades [22]. IP3RyRs have
been identified in trypanosomes and paramecium [12, 23]. However, they are localized
in vacuoles that store Ca2+ and function in osmoregulation. Interestingly, IP3RyRs
have not been identified thus far in other protozoans such as Plasmodium sp. or Giardia
sp. Yeast also lacks IP3RyRs, and intracellular Ca2+ levels are regulated via a vacuolar
transient receptor potential channel that functions in osmoregulation [24]. Hence,
IP3 may function in osmoregulation in some protozoans and perhaps evolved functions
that are specific to the biology of these organisms.
T. brucei inositol polyphosphate multikinase (IPMK) phosphorylates IP3 and generates
inositol tetra (IP4) and pentakisphosphate (IP5) [6, 13], which are further phosphorylated
into inositol hexakisphosphate (IP6) and inositol pyrophosphates (PP-IPs) [18]. These
IPs play essential roles in trypanosomes, as evidenced by the finding that knockdown
or catalytic mutations of T. brucei IPMK affect survival, development, and metabolism
(discussed below) [6, 13] (Table 1). IPMK inhibitors also affect T. cruzi amastigote
proliferation [13], and knockdown of T. cruzi IP3RyR affects growth, survival, and
differentiation [19]. The molecular basis underlying IP regulatory function in T.
brucei is likely to function analogous to their yeast and metazoan counterparts, i.e.,
by interacting with proteins and thus regulating protein activity, interactions, or
localization [16, 25–27]. T. brucei has several proteins that bind to IP3 or IP4 [6],
most of which function in metabolism, protein synthesis and turnover, motility, and
signal transduction [6]. The control of IP phosphorylation, and thus their association
with proteins, provides a reversible and fast regulatory mechanism to control protein
function. The characteristics of this system may be essential to regulate cellular
processes in response to rapid environmental or physiological changes during parasite
development and infection.
Nuclear PIs: Transcriptional control of variant surface glycoprotein genes and antigenic
variation
T. brucei expresses a homogeneous surface coat of variant surface glycoproteins (VSGs)
and periodically switches its expression to escape host antibody recognition in a
process known as antigenic variation. This parasite selectively expresses one out
of hundreds of VSG genes, which is transcribed from one of about 20 telomeric expression
sites (ESs). T. brucei changes VSG expression by transcriptional switch between ESs
or by VSG gene recombination (reviewed in [4]). The control of VSG-exclusive expression
and switching entails a regulatory system that includes nuclear proteins, e.g., chromatin
regulatory proteins, nuclear lamina proteins, and nonnuclear proteins [4]. Phosphatidylinositol
phosphate 5-kinase (PIP5K) and PLC, both of which localize in the plasma membrane
inner leaflet and endosomal compartments, regulate VSG allelic exclusion and switching
[7]. Knockdown of PIP5K results in simultaneous transcription of all telomeric ES
VSG genes. Reexpression of PIP5K resumes VSG-exclusive expression but results in switching
of the VSG gene expressed by either transcriptional or recombination mechanisms. Moreover,
overexpression of PLC, but not a mutant catalytic inactive version of PLC, results
in transcription of silent VSG genes [7]. The involvement of these proteins in VSG
silencing and switching is suggestive of a signal transduction system that is reactive
to cellular changes, perhaps via external stimuli or inherent to developmental processes.
How such a system regulates silencing and switching of VSG genes is yet unclear, but
it might involve the control of PIPs subcellular fluxes and levels. T. brucei expresses
a nuclear phosphatidylinositol phosphate 5-phosphatase (PIP5Pase) enzyme that also
controls silencing of telomeric and subtelomeric VSG genes [5]. PIP5Pase associates
with repressor-activator protein 1 (RAP1) within a 0.9 megadalton protein complex,
which also includes protein kinases, phosphatases, chromatin regulatory proteins,
and nuclear pore proteins [5, 7]. PIP5Pase regulation of VSG silencing revolves around
the control of phosphatidylinositol 3,4,5-triphosphate (PIP3) levels. PIP5Pase dephosphorylates
PIP3 and prevents this metabolite from interacting with RAP1. Catalytic mutations
of PIP5Pase that inhibit PIP3 dephosphorylation but do not disrupt PIP5Pase protein
complex integrity result in transcription of silent VSG genes, indicating that PIP5Pase
activity is essential for VSG silencing [5]. In the current model, PIP5Pase dephosphorylation
of PIP3 prevents the binding of this metabolite to RAP1, which preserves the association
of RAP1 (and likely other proteins within the complex) with ES chromatin and represses
transcription of VSG genes (Fig 1D). Conversely, the inactivation of PIP5Pase results
in PIP3 binding by RAP1, which affects RAP1 association with ES chromatin and thus
chromatin organization and results in VSG gene transcription [5]. Hence, PIPs play
a key role in the mechanisms that control VSG-exclusive expression and switching and
provide a hint as to how VSG regulation might be integrated with regulatory and signal
transduction processes.
IP regulation of energy metabolism and development
T. brucei life stage development entails dramatic changes in energy metabolism. Mammalian
infective bloodstream forms (BFs) switch energy metabolism from glycolysis, which
occurs in glycosomes (peroxisomes-like organelles), to a more complex metabolism that
includes mitochondrial oxidative phosphorylation in the insect stage procyclic forms
(PFs) [28]. This metabolic regulation involves IPs and is coregulated with parasite
development [6]. The knockdown or catalytic mutation of IPMK in BFs results in the
activation of oxidative phosphorylation, which is accompanied by a 20-fold increase
in parasite development from BFs to PFs [6]. After IPMK knockdown, BFs up-regulate
the expression of genes encoding mitochondrial respiratory complexes, some of which
are only expressed in PFs, thus producing a functional respiratory chain that generates
ATP. The molecular control of T. brucei metabolic switch likely involves IPMK substrates
and products, e.g., IP3 and IP4, which interact with proteins involved in metabolism,
protein synthesis and degradation, and signal transduction [6]. IPMK inactivation
also affects the expression of RNA-binding proteins (RBPs), some of which control
the expression of stage-specific genes and are involved in parasite life stage development
[6, 29, 30]. Hence, IPMKs play a key role in the regulation of T. brucei metabolic
switch and development.
The IPMK role in energy metabolism is conserved among T. brucei, yeast, and mammalian
cells. IPMK controls the metabolic switch from oxidative phosphorylation to glycolysis
in yeast and to aerobic glycolysis in cancer cells [17], known as the Warburg effect.
In human cells, IPMK regulates cell metabolism independent of its catalytic activity.
IPMK interacts with 5' adenosine monophosphate-activated protein kinase (AMPK) and
mammalian target of rapamycin (mTOR) and regulates glucose and amino acid signaling
[31, 32]. AMPK and target of rapamycin 4 (TOR4) complexes are also involved in T.
brucei metabolism and development [2, 3], but it is unknown if they associate with
T. brucei IPMK. Moreover, the control of energy metabolism in T. brucei depends on
IPMK catalytic activity and thus on IPs [6]. In yeast, IPs control the activity of
transcription factors such as glycolytic genes transcriptional activator (GCR1) and
ArgR-Mcm1 transcription complex, which are involved in the expression of glycolysis
and amino acid metabolism genes [17, 25]. However, unlike in yeast, T. brucei controls
gene expression posttranscriptionally by regulation of RNA stability or translation,
in which RBPs play a critical role. Hence, IPMK roles in metabolic regulation likely
originated early in eukaryotes, but its regulatory mechanisms diversified among eukaryote
organisms. In T. brucei, the switch in energy metabolism entails a regulatory system
that involves IPs and posttranscriptional control of gene expression [6].
Conclusion and perspectives
PIPs and IPs have regulatory functions in T. brucei that are conserved with other
eukaryotes [9, 10]. However, PIPs and IPs have also diversified in function to control
specialized processes such as antigenic variation in T. brucei [5, 7]. Regulation
of this process might be conserved in other protozoan pathogens such as Plasmodium
and Giardia, which also employ antigenic variation. The finding that PIP5K and PLC
control VSG expression and switching raises the question of whether a signal transduction
system is involved in the control of antigenic variation. VSG switching is thought
to occur stochastically and perhaps initiated by events that lead to DNA break and
repair [33]. An alternative hypothesis is that initiation of VSG switching occurs
by activation of a signal transduction system that involves PIPs. This process may
happen in addition to PIPs function in the control of VSG-exclusive expression via
regulation of protein association with telomeric ES DNA [5, 7]. In contrast to PIP
roles in VSG expression, the regulation of energy metabolism by IPMK while sharing
conserved features among eukaryotes has diversified in the mechanisms of regulation,
with T. brucei relying heavily on posttranscriptional control of gene expression [6,
17]. These are a few examples of many processes that are likely regulated by PIPs
and IPs in T. brucei and in related pathogens.
There are many fundamental questions related to PIP and IP functions that remain unknown.
One such question is whether PIPs and IPs are transported from cytoplasmic organelles
to the nucleus or are synthesized in the nucleus. The identification of PIP5Pase in
the nucleus of T. brucei is an indication that the interconversion of these metabolites
occurs in the nucleus [5, 7], and PIP and IP interconversions have also been detected
in the nucleus of yeast and mammalian cells [25, 34, 35]. Nevertheless, the molecular
machinery that are involved in PIPs and IPs nuclear synthesis or their transport is
unknown. Furthermore, the crosstalk between PIPs and IPs with other cell signaling
and regulatory systems to control specific processes, e.g., energy generation or cell
development, is also poorly understood. Answer to these questions may require the
identification of signaling receptors, effector proteins, and proteins that link PIPs
and IPs with distinct signaling pathways, e.g., cyclic adenosine monophosphate (cAMP)
signaling, and the input and output of these pathways. Although many proteins have
been shown to bind PIPs and IPs [6, 27], there are only a few protein domains that
are known to interact with these metabolites [36]. In addition, interactions of PIPs
with RNAs have been sparingly identified [37]. It is unknown how widespread PIP and
RNA interactions are in eukaryotes, but trypanosomes, which rely on posttranscriptional
control processes to regulate gene expression, may be an excellent model to study
the potential functions of PIPs and RNA interactions. Understanding the role of PIP
and IP signaling and regulation in T. brucei may help us to gain insights on the function,
regulation, and evolution of signaling pathways in pathogens and in more complex multicellular
eukaryotes.