Download Gene expression in Plasmodium: from gametocytes to sporozoites

Gene expression in Plasmodium
Gene expression in Plasmodium: from
gametocytes to sporozoites
Disruption of the parasite life cycle in the vector, such as with
transmission blocking vaccines (TBVs), is a strategy that offers much
promise. TBVs use antigens unique to the mosquito forms of Plasmodium
to stimulate the production of antibodies in the vertebrate host. When the
female mosquito takes an infectious blood meal, it also ingests antibodies
that interfere with parasite development, thus preventing transmission to
another individual. Several Plasmodium proteins have been tested as
TBV candidates (Moreira et al., 2004).
The malaria parasite possesses several lectin-like proteins capable
of binding the glycoconjugates that decorate the surface of eukaryotic
cells, including hepatocytes, erythrocytes and endothelial cells to the
parasite surface (Itzstein et al., 2008).
1. Lectins and genes expressed by gametocytes and gametes
Plasmodium falciparum sexual differentiation is divided into five
morphological stages (I–V) that may last between 8 and 17 days inside
the red blood cells. These morphological changes are accompanied by
distinct patterns of sexual stage-specific gene expression. Pfs16 is
expressed within 24 h following the invasion of a red blood cell and is the
earliest known sexual stage-specific gene. The protein is synthesized
throughout gametocytogenesis, in male and female gametocytes, and
localises to the parasitophorous vacuole membrane of the parasites
(Bruce et al., 1994).
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Gene expression in Plasmodium
Plasmodium life cycle and temporal patterns of gene expression. The
solid lines indicate parasite stages at which protein was detected, and the
dashed lines indicate parasite stages at which mRNA, but not the
corresponding protein, was detected (Moreira et al., 2004).
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Gene expression in Plasmodium
Knockout of the Pfs16 gene led to a four- to five-fold reduction in
the number of gametocytes produced, although the morphology of the
cells did not seem to be altered. Moreover, knockout male gametocytes
were unable to exflagellate and these parasites were not infectious to
mosquitoes. The results suggest that Pfs16 is not essential for sexual
development, but may be required for optimal production of sexual
parasites (Kongkasuriyachai et al., 2004).
Pfg27 is expressed at about 30 h post-erythrocytic invasion, by
both gametocytes sexes and is distributed throughout the cell cytoplasm.
Pfg27 knockout parasites seem to be committed to sexual differentiation
since they synthesise Pfs16, but fail to develop further, resulting in
parasites that are vacuolated and eventually die. Sharma et al. (2003)
speculated that Pfg27 may enable the formation of a multi-protein
complex that mediates transduction of external signals and leads to the
interaction of Pfg27 with specific RNAs.
Pfs48/45 expressed from stage III male and female gametocytes
until zygote, and the proteins, localised on the surface of male and female
gametes and of zygotes. Disruption of this gene demonstrated a central
role in male gamete fertility. Gametocyte production and differentiation
into gametes was unaffected in knockout parasites but male gametes were
unable to adhere to and penetrate female gametes, strongly decreasing
fertilization and zygote formation (Moreira et al., 2004).
A calcium dependent protein kinase, CDPK4, predominantly
expressed in male gametocytes, was identified. The xanthurenic acid, a
small mosquito molecule known to induce gametogenesis, triggers a rapid
rise in cytosolic calcium activating this kinase. Knockout of the CDPK4
gene specifically hindered male gametogenesis by inhibiting DNA
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Gene expression in Plasmodium
replication and mitotic spindle formation, suggesting that CDPK4 acts as
a regulator of cell cycle progression in the microgametocyte (Billker et
al., 2004).
2. Lectins and genes expressed by zygotes, ookinetes and
oocyst
Following fertilization, the resulting zygote differentiates into a motile
ookinete that around 24 h traverses the peritrophic matrix and midgut
epithelium, leading to the formation of an oocyst (Moreira et al., 2004).
P25 and P28 are major proteins on the surface of zygotes and
ookinetes. Their protein synthesis begins 30 min after the formation of
female gametes in the mosquito and continues until late oocyst (Moreira
et al., 2004). Knockout experiments demonstrated that P25 and P28 have
redundant functions since single gene disruption does not affect parasite
development and caused only a slight decrease in oocyte formation. By
contrast, double knockouts gave rise to considerably fewer ookinetes and
virtually no oocysts were formed. It was concluded that the double
knockout had defects in both penetration and oocyst formation. Ookinetes
from the double knockouts were also more susceptible to trypsin (Roditi
and Liniger, 2002).
Four ookinete micronemal proteins have been characterized.
Chitinase, von Willebrand factor A domainrelated protein (WARP) and
secreted ookinete adhesive protein (SOAP) are soluble proteins, and the
circumsporozoite- and TRAP-related protein (CTRP) is membrane
associated (Moreira et al., 2004).
Chitinase genes were identified in all Plasmodium species tested
and are expressed in ookinetes (Tsuboi et al., 2003). Targeted disruption
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Gene expression in Plasmodium
of Pfcth1 (from P. falciparum) and Pbcth1 (from P. berghei) resulted in a
significant decrease in the number of parasites that traversed the midgut
and formed oocysts, showing the importance of this enzyme for the
penetration of the chitin containing peritrophic matrix (Vinetz et al.,
2000).
The CTRP gene has been characterised in P. berghei and P.
falciparum. CTRP synthesis is upregulated at around 10 h after
fertilization, when zygotes begin transformation to ookinetes. While the
mRNA can be detected at low levels in gametocytes and peaks at the
ookinete stage, the protein can only be detected from the retort stage
(transition form from zygote to ookinete) onward. The extracellular
region of this protein contains six von Willebrand factor type A-related
and seven human thrombospondin type I-related adhesive domains,
known to participate in cell–cell and/or cell–matrix interactions. This
domain structure is shared with the Plasmodium thrombospondin-related
anonymous protein (TRAP), expressed in sporozoites, which is protein
that has been implicated in gliding motility and in cell invasion. CTRP
knockout parasites developed normal numbers of gametes and ookinetes
in vitro, although the latter were less motile than wild-type parasites.
Ookinetes were also able to develop in the midguts of two mosquito
vectors, Anopheles gambiense and Anopheles stephensi, but, in contrast to
wild-type ookinetes, these did not penetrate the epithelium and did not
form oocysts. When the CTRP gene was disrupted in P. falciparum,
ookinete development was again unaffected, and there was a complete
loss of oocyst production, suggested that this protein could play a dual
function in motility and binding to the epithelial cells (Roditi and
Liniger, 2002).
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Gene expression in Plasmodium
WARP is a soluble protein that contains a von Willebrand factor
A-like domain and is expressed in late ookinetes and early oocysts. It has
been suggested that WARP plays a role in adhesion. During midgut
invasion. WARP could mediate ookinete attachment to the mosquito
midgut, and during ookinete to oocyst differentiation it could mediate
interactions with the mosquito basal lamina (Yuda et al., 2001; Abraham
et al., 2004).
SOAP expressed in ookinetes and early oocysts. Knockout of the
PbSOAP gene (from P. berghei) led to a reduction in the number of
oocysts formed but oocysts that did form developed normally and the
resulting sporozoites were infective to mice, suggesting that PbSOAP is
mainly required for midgut invasion (Arrighi and Hurd, 2002).
3. Lectins and Genes expressed by sporozoites
Upon maturation, each oocyst releases into the hemolymph thousands of
sporozoites, which in turn traverse the salivary gland epithelium to lodge
in the salivary gland lumen. Once injected into a vertebrate host,
sporozoites rapidly invade liver cells where they develop into forms
capable of invading red blood cells. Inoculation of mammals, including
humans, with sporozoites attenuated by radiation led to malaria protection
and the identification of immune-protective sporozoite antigens has been
the object of study in many laboratories (Moreira et al., 2004).
The best-studied of the proteins are the circumsporozoite protein
(CS) and the thrombospondin-related anonymous protein (TRAP). Both
proteins have been identified in all Plasmodium species. These proteins
are also involved in salivary gland and hepatocyte invasion (Sultan et al.,
1997).
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Gene expression in Plasmodium
CS covers the entire sporozoite surface. Whereas CS transcription
starts at the early oocyst stage and peaks in salivary gland sporozoites, the
protein is first detected only in mid to late oocysts (however, small
amounts of protein must be synthesized earlier. Sporozoite binding to
mosquito salivary glands and attachment to heparan sulfate proteoglycans
of the mammalian liver is mediated by specific CS motifs (Roditi and
Liniger, 2002). CS is also involved in gliding motility, being secreted at
the anterior pole, translocated along the sporozoite surface and released
on the substrate at the posterior pole. CS knockout parasites grow
normally in the mammalian host but development in the mosquito is
arrested at the early oocyst stage. It appears that CS is involved in
sporozoite morphogenesis inside oocysts. The extent of development of
the inner membranes and associated microtubules underneath the oocyst
outer membrane is directly correlated with the amount of CS protein
synthesised by the sporozoite (Thathy et al., 2002).
The second major sporozoite protein, TRAP, is located on the
surface of midgut and salivary gland sporozoites and in micronemes.
Transcription and translation of TRAP starts in late oocysts, after
completion of sporozoite morphogenesis. The TRAP protein contains two
distinct adhesive domains (TSP and A) that were implicated in salivary
gland invasion. The domains have similarity with the type I repeat of
thrombospondin (TSP) and the von Willebrand factor type A-domain.
Sporozoites carrying a mutation in the A domain of the PfTRAP are still
motile while sporozoites with a TSP motif deletion have no gliding
motility. Neither of the mutants can invade salivary glands. Thus, TRAP
is important for motility and salivary gland invasion. A recent structural
and functional study of the TRAP adhesive domains demonstrated that
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Gene expression in Plasmodium
this protein also interacts with multiple receptors during the hepatocyte
invasion process (Akhouri et al., 2004).
Claudianos et al. (2002) searched the P. falciparum genome for
genes encoding scavenger receptor cysteine-rich domains (SRCR) and
identified a single putative gene, PfSR. SRCR domains have been
implicated in immune recognition/activation and lipid/protein adhesion.
PCR analysis of the P. berghei homolog (PbSR) showed that its transcript
is detected in asexual and sexual stages of parasite differentiation.
However, the protein was only detected in midgut- and salivary glandsporozoites, indicating that transcription and translation are uncoupled.
PbSR knockout parasites produced normal numbers of oocysts but no
sporozoites developed. Like all known proteins containing SRCR
domains, PbSR has structural features indicating that it may play a
protective role against mosquito immune factors that can affect sporozoite
formation. As previously reported for CS and TRAP, SR protein could
also play a role in liver infection since it is present in the salivary gland
sporozoites (Moreira et al., 2004).
Transcripts of MAEBL were detected among the expressed
sequence tags (ESTs). Expression of this protein was first identified in
erythrocytic forms of P. yoelii and P. berghei where it is located in the
rhoptry organelles of merozoites. MAEBL binds to red blood cells
indicating that it plays a role in red blood cell invasion by merozoites.
Disruption of this gene revealing that MAEBL is essential for sporozoite
invasion of the salivary glands. However, the disruption did not affect
sporozoite motility and infectivity to the vertebrate host (Kariu et al.,
2002).
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Gene expression in Plasmodium
AMA-1 (Anti mammalian antigen-1) has been extensively studied
in asexual stages, where it functions in merozoite invasion of
erythrocytes. Srinivasan et al. (2004) found that AMA-1 is also
expressed in mosquito stages. The mRNA accumulates to high levels in
late oocysts and remains abundant in salivary gland sporozoites. By
contrast, the AMA-1 protein is undetectable in late oocysts or sporozoites
before salivary gland invasion, but is abundant on the surface of salivary
gland sporozoites. Thus, salivary gland invasion appears to activate the
translation of AMA-1 mRNA. That the protein is synthesized only after
salivary gland invasion and being located on the sporozoite surface
suggests that it functions in invasion of the vertebrate liver. Indeed, Silvie
et al. (2004) have shown that anti-AMA-1 antibodies inhibit P.
falciparum sporozoites invasion of hepatocytes.
The secreted protein with altered thrombospondin repeat (SPATR)
is an EST identified by Kappe et al. (2001) that was considered a
potential sporozoite invasion ligand. Considering that CS protein and
TRAP carry the same type 1 thrombospodin repeat and that both proteins
have important roles in sporozoite motility, binding and invasion to the
host-cell, Kappe et al. (2001) suggested that SPATR may have a similar
function.
The sporozoite microneme protein essential for cell traversal
(SPECT) was identified by screening an EST database of P. berghei
salivary gland sporozoites. SPECT is a 22 kDa micronemal protein found
specifically in salivary gland sporozoites. Targeted disruption of the
SPECT gene highly inhibited sporozoite infectivity to the liver (Ishino et
al., 2004).
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Gene expression in Plasmodium
Proteomic analysis showed that members of the var or P.
falciparum erythrocyte membrane protein 1 (PfEMP1) family and rifin
genes are expressed in P. falciparum salivary gland sporozoites. The
corresponding proteins were originally thought to be expressed only on
the surface of infected red blood cells, where they are believed to function
in immune evasion. Expression of var and rifin proteins in sporozoites
may be related to parasite survival in mosquitoes and vertebrate hosts
even though sporozoites do not undergo antigenic ‘switching’ as in
asexual stages (Florens et al., 2002).
Plasmodium sporozoites, like all invasive forms of the
apicomplexan parasites, possess typical apical organelles that secrete
proteins involved in the motility and host-cell invasion. These processes
are dependent on the actin/ myosin system and have been detected in
ookinete and sporozoite forms. An unconventional myosin was identified
in sporozoite forms of P. berghei and P. yoelii being co-localised with
TRAP (Matuschewski et al., 2001). As discussed above, TRAP is also
essential for sporozoite motility and infectivity. It was suggested that
TRAP connects the parasite actin–myosin system with external substrates
delivering a specific signal upon binding to host ligands. Another myosin,
named ‘myosin-A’, was also found in sporozoites and ookinetes,
supporting the idea that myosin is used by all the different invasive forms
of malaria parasites and involved in gliding motility (Matuschewski et
al., 2001).
Conclusion
Considering that investigation of the molecular events that guide
development of the malaria parasite in the mosquito started in earnest
only a few years ago, progress made to date has been impressive. One
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Gene expression in Plasmodium
example is the engineering of transgenic Anopheles mosquitoes
expressing effector molecules that interfere with parasite development.
While much work still lies ahead, the prospects are bright. The
understanding of how gametes are formed and how the parasite manages
to cycle through the mosquito while evading its immune defenses
promises to lead to the development of new approaches to malaria control
(Moreira et al., 2004).
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