Innate immunity in the malaria vector Anopheles gambiae: comparative and functional genomics
European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
* Author for correspondence (e-mail: dg-office{at}embl.de)
Accepted 29 April 2004
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Summary |
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Key words: malaria, innate immunity, Anopheles gambiae, genomics, disease control, pattern recognition receptor
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Introduction |
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In insects, the peritrophic membrane (PM) of the midgut, the cuticle of the
exoskeleton and the lining of the tracheal respiratory system constitute
physical barriers against invaders. The PM is a sleeve-like extracellular
layer surrounding the food bolus and is composed of chitin, proteins and
proteoglycans (Wang and Granados,
2001). Ingested ookinetes of the malaria parasite
Plasmodium must penetrate the midgut wall of the Anopheles
mosquito vector to develop further into oocysts on the haemocoel basal side of
the midgut epithelium (Fig. 1).
This process is facilitated by the secretion of chitinases that disrupt the PM
chitin (Huber et al., 1991
;
Langer et al., 2000
). Midgut
invasion was completely blocked when mosquitoes were fed on infected blood
containing the chitinase inhibitor allosamidin
(Shahabuddin et al., 1993
).
Similarly, knockout of the Plasmodium falciparum chitinase gene
induced a marked reduction in the number of developing oocysts in the midguts
of Anopheles freeborni mosquitoes
(Tsai et al., 2001
).
Microorganisms that successfully overcome any of these physical barriers
encounter an array of host innate immune responses within the underlying
epithelia or systemically in internal immune tissues such as haemocytes
(insect blood cells) and the fat body (liver analogue), which are known to
release immune effectors into the open circulatory system, the haemolymph (the
insect blood). Examples of such responses are phagocytosis, secretion of
antimicrobial peptides, nodule formation, agglutination, encapsulation and
melanization.
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Studies on Drosophila innate immunity have had a major impact on
this field, both in vertebrates and invertebrates, leading to key discoveries
and fundamental concepts on how organisms efficiently fight pathogens even in
the absence of the clonal system of recognition that is central to adaptive
immunity (Hoffmann, 2003;
Hoffmann and Reichhart, 2002
).
What made Drosophila tractable for such studies was the powerful
genetic and molecular genetic tools available in the fruitfly, including a
fully sequenced genome. The mosquito Anopheles gambiae, the major
vector of malaria in Africa, also became a suitable model for the study of
innate immunity recently, when its genome was sequenced
(Holt et al., 2002
),
accompanied by comparative genomic analysis with Drosophila
(Zdobnov et al., 2002
) and
the establishment of powerful tools for gene discovery
(Dimopoulos et al., 2002
),
functional analysis (Blandin et al.,
2002
; Levashina et al.,
2001
) and transgenesis
(Grossman et al., 2001
). As
the most important vector for transmission of the malaria parasite, A.
gambiae offers the advantage of assessing immune reactions in a species
of major importance to human health. A. gambiae mounts efficient
local and systemic immune responses against Plasmodium infection
(Dimopoulos et al., 1997
,
1998
;
Richman et al., 1997
;
Fig. 1). However, in spite of
the hostile environment encountered and major losses in parasite numbers
inside the vector, some parasite species or strains successfully complete
their sexual life cycle, indicating that they have found ways to escape or
subvert to some extent the vector immune responses. Other
vectorparasite combinations are either poor (Plasmodium
gallinaceum/Anopheles stephensi) or incompatible (Plasmodium
berghei/Aedes aegypti), suggesting that key molecular and
cellular interactions are a prerequisite for a vectorparasite system to
become established and subsequently co-evolve
(Alavi et al., 2003
). In this
review, we describe our current knowledge of A. gambiae innate
immunity and its impact on Plasmodium development.
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Recognition of non-self |
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GNBPs were first described in Bombyx mori
(Lee et al., 1996) and share
significant sequence similarity with the catalytic region of bacterial
ß-1,3- and ß-1,3,1,4-glucanases. BmGNBP binds strongly to
the surface of Gram-negative bacteria and shows pronounced transcriptional
upregulation following bacterial challenge
(Lee et al., 1996
). The
Drosophila GNBP1 binds with high affinity to LPS and
ß-1,3-glucan (Kim et al.,
2000
) and, in concert with PGRP-SA, activates the Toll pathway
upon infection with Gram-positive bacteria
(Gobert et al., 2003
). In
A. gambiae, six putative GNBPs have been identified
(Christophides et al., 2002
).
Among them, GNBPB1 and GNBPA1 are upregulated following
Plasmodium infection, while only GNBPB1 is responsive to
bacteria (Christophides et al.,
2002
; Dimopoulos et al.,
2002
). Other putative Anopheles PRRs include the
thioester-containing proteins (TEPs), leucine-rich immune proteins (LRIMs) and
C-type lectins (CTLs). Members of these protein families were recently
implicated in the regulation of Plasmodium development in the
mosquito vector and are discussed later in this review.
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Signal modulation and transduction |
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Melanization requires the enzymatic processing of inactive PPO to active
phenoloxidases (PO) by activating serine proteases, referred to in the
literature as prophenol-activating proteinase (PAP) or
prophenoloxidase-activating enzyme (PPAE). In the tobacco hornworm Manduca
sexta, several PAPs have been described and shown biochemically to be
involved in PPO activation: PAP1 has one clip-domain
(Jiang et al., 1998) while
PAP2 (Jiang et al., 2003a
) and
PAP3 (Jiang et al., 2003b
)
contain two clip-domains. Interestingly, the Manduca PAPs require
non-proteolytic serine protease homologues as cofactors for the efficient
activation of PPO (Jiang et al.,
2003a
,b
;
Yu et al., 2003
). Two A.
gambiae CLIPs (CLIPB14 and CLIPB15) were found to be responsive to
bacterial and Plasmodium infections: CLIPB14 showed persistent
upregulation in Plasmodium-infected mosquitoes while CLIPB15 showed
only transient upregulation, precisely during midgut invasion
(Christophides et al., 2002
).
The role of these two genes in the activation of PPO has been demonstrated
recently (J. Volz, unpublished data).
Signal amplification by the serine protease cascade is under tight
regulation by serpins (serine protease inhibitors), which inhibit serine
proteases by acting as irreversible suicide substrates that covalently bind to
the active centre of the enzyme. The Drosophila serpin Spn43Ac
(Necrotic) downregulates the Toll pathway in response to fungal infections by
inhibiting cleavage of the Toll receptor ligand, the cytokine-like polypeptide
Spaetzle (Levashina et al.,
1999). The involvement of serpins in the regulation of
melanization has been described in D. melanogaster
(De Gregorio et al., 2002
;
Ligoxygakis et al., 2002b
),
M. sexta (Zhu et al.,
2003
) and A. gambiae (K. Michel, unpublished data). A
recently characterized Anopheles serpin, SRPN10, encodes four
alternatively spliced inhibitory isoforms
(Danielli et al., 2003
).
Interestingly, two of these forms are specifically upregulated in female
mosquitoes in response to midgut invasion by P. berghei ookinetes,
making SRPN10 an excellent cell-autonomous marker of invasion (A. Danielli, T.
G. Loukeris and C. Barillas-Mury, unpublished data).
Immune signalling pathways
Immune signalling pathways transmit the signal originating from
PAMP-associated PRRs to the effector genes. In a now classical series of
studies, two signal transduction pathways, the Toll and IMD pathways
(Fig. 2), have been identified
in Drosophila and linked to innate immunity
(Hoffmann, 2003). Fungal or
Gram-positive bacterial infections activate the Toll pathway by inducing the
proteolytic cleavage of Spaetzle, which binds directly to and activates the
transmembrane receptor Toll (Weber et
al., 2003
). Hence, Toll does not appear to be a direct sensor of
microbial compounds, unlike the mammalian Toll-like receptors (TLRs) that
recognize and bind to several microbial ligands
(Takeda et al., 2003
). Toll
has an intracytoplasmic TIR (Toll, IL-1R) domain, which upon activation
recruits three death domain proteins, MyD88, Tube and Pelle, the first two of
which are considered as adaptor proteins. The Toll receptoradaptor
complex signals to two cytoplasmic Rel/NF
B transcription factors,
Dorsal and Dif, causing their dissociation from Cactus, an ankyrin repeat
inhibitory protein, and subsequent translocation to the nucleus where they
activate the transcription of AMPs. Whereas Dif has a crucial role in the
immune response (Ip et al.,
1993
; Petersen et al.,
1995
), Dorsal is believed to be mainly engaged in transcriptional
activation of genes involved in the dorsoventral patterning
(Morisato and Anderson, 1995
).
Interestingly, no orthologue of Dif was found in the Anopheles
genome. Gambif1 (now called REL1), the mosquito orthologue of Dorsal, has been
previously characterized and shown to translocate to the nucleus following
bacterial but not Plasmodium infection
(Barillas-Mury et al., 1996
).
Ten Toll and six Spaetzle-like proteins have been identified in the genome of
A. gambiae, but their phylogenetic relationships with the respective
Drosophila homologues are unclear
(Christophides et al., 2002
).
However, identification of the mosquito orthologues of MyD88, Tube and Pelle
indicates that the Toll pathway in the mosquito is at least partially
conserved (Christophides et al.,
2002
).
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The second immune signalling pathway in Drosophila, the IMD
pathway, is activated following Gram-negative bacterial infection leading to
the cleavage of another Rel/NFB family protein, Relish, through the
recently proposed proteolytic action of the caspase Dredd
(Stoven et al., 2003
); this
causes the release of the Rel-homology domain of Relish from its inhibitory
carboxy-terminal ankyrin domain. The transmembrane receptor of the IMD pathway
is as yet unclear; however, a number of studies point to a role for PGRP-LC in
this process (Choe et al.,
2002
; Gottar et al.,
2002
). Intracellular activation of the pathway commences with
recruitment of IMD, a death domain protein sharing similarities with the
mammalian TNF-
receptor interacting protein, RIP. The sequence of
events between IMD activation and the cleavage of Relish is not fully
characterized; however, genetic data point to several other factors downstream
of IMD, including the protein kinase dTAK1, another death-domain protein,
dFADD, and the Drosophila homologues of the mammalian signalosome
equivalent comprising IKK-ß and IKK-
. Interestingly, all the
aforementioned components of the IMD pathway are conserved in
Anopheles (Christophides et al.,
2002
), and preliminary data indicate that REL2, the
Anopheles orthologue of Relish, is indeed involved in anti-bacterial
defence (G. K. Christophides, unpublished data). The Ae. aegypti
Relish gene has three alternatively spliced transcripts encoding three
different proteins: Relish, I
B-type, which lacks the Rel-homology
domain, and the Rel-type in which the amino-terminal transactivation domain
and the carboxy-terminal ankyrin repeats are missing
(Shin et al., 2002
). The
involvement of Aedes Relish in the regulation of immune response to
bacterial challenge has been shown using transgenic mosquitoes carrying the
Rel-type transgene driven by the bloodmeal-inducible vitellogenin promoter
(Shin et al., 2003a
). In these
mosquitoes, the overexpression of Rel-type transgene following a blood meal
resulted in severely reduced expression levels of defensin and cecropin genes
and a strong susceptibility to Gram-negative bacterial infections. The
interference of Rel-type protein with endogenous Relish was suggested to
involve competitive binding to the
B motif.
Little is known about the role of the JNK and JAK/STAT pathways in
antimicrobial defence in insects. The Drosophila JAK/STAT pathway has
one STAT component and is involved in many developmental processes
(Luo and Dearolf, 2001). A
recent study based on microarray analysis on Drosophila cell lines
revealed that the IMD pathway branches downstream of dTAK1: one branch
controlling the synthesis of antimicrobial peptides through Relish, and the
other the synthesis of cytoskeletal proteins through the JAK/STAT
(Boutros et al., 2002
). Based
on these data, a close link between cytoskeletal remodelling and antimicrobial
defence was suggested (Boutros et al.,
2002
). Two members of the STAT family (STAT1 and STAT2) have been
identified in the mosquito genome
(Christophides et al., 2002
).
The observed STAT1 (previously called AgSTAT) translocation into the
nucleus of mosquito fat body cells following bacterial infection has provided
the first evidence for the involvement of insect STATs in immune defence
(Barillas-Mury et al.,
1999
).
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Pathogen elimination |
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Phagocytosis
Phagocytosis is a hallmark of the classical cellular immune responses of
insects whereby haemocytes engulf target pathogens but also apoptotic bodies.
Three types of haemocytes have been characterized in Drosophila: the
plasmatocytes that are responsible for the disposal of microorganisms and
apoptotic cells, the lamellocytes that encapsulate large invaders, and the
crystal cells that are involved in melanization
(Meister and Lagueux, 2003).
In the yellow fever mosquito, Ae. aegypti, four different types of
haemocytes have been distinguished: the granulocytes, the oenocytoids, the
adipohaemocytes and the thrombocytoids
(Hillyer and Christensen,
2002
). Phagocytosis in Drosophila is usually receptor
mediated, involving either soluble or membrane-bound PRRs, including PGRP-LC,
which is involved in the phagocytosis of Gram-negative bacteria
(Ramet et al., 2002
), and
Croquemort, a membrane-bound receptor mediating phagocytosis of apoptotic
corpses (Franc et al., 1996
).
A soluble thioester-containing protein, TEP1, identified in A.
gambiae, acts as a complement-like opsonin promoting the phagocytosis of
Gram-negative bacteria in a mosquito haemocyte-like cell line
(Levashina et al., 2001
). TEP1
activation seems to result from its infection-inducible proteolytic cleavage
and exposure of the highly reactive thioester bond, apparently involved in a
nucleophilic attack leading to the covalent binding of TEP1 to the surface of
microorganisms.
Cellular encapsulation
The mechanisms promoting cellular encapsulation in insects are not well
understood, and only a few examples of this innate cellular reaction have been
reported. Encapsulation is a process by which insect lamellocytes form a
multilayered capsule around large invaders such as parasitoids in the
haemocoel, resulting in their isolation, immobilization and subsequent killing
by asphyxiation, oxidation or melanization
(Gotz, 1986).
Drosophila larvae encapsulate and then melanize the eggs of a
parasitoid wasp in their haemocoel through the concerted action of
lamellocytes and crystal cells (Lanot et
al., 2001
; Sorrentino et al.,
2002
). Hemese, a recently identified transmembrane receptor, is
expressed on the surface of all Drosophila haemocytes and acts as a
negative regulator of the encapsulation response
(Kurucz et al., 2003
).
Knockout of Hemese stimulates the proliferation of lamellocytes following
parasitoid infection, resulting in an enhanced cellular response
(Kurucz et al., 2003
). The
mechanisms through which lamellocytes are alerted to the presence of a large
invader and the signals involved in their proliferation remain unknown.
Melanization
Melanization is a prime humoral immune reaction of insects, being involved
in wound healing and sequestration of invaders in a dense melanin coat. In
contrast to encapsulation, it does not require the direct involvement of
haemocytes (Soderhall and Cerenius,
1998). Melanization requires the proteolytic activation of the
inactive PPO zymogens to the active POs, a step tightly regulated, as
described above, by the balanced action of CLIPs and their inhibitors,
serpins. POs oxidize phenolic substances such as tyrosine, DOPA and dopamine
to melanine and serve several tasks including wound healing, cuticle
pigmentation and sclerotization, and melanization of invading pathogens
(Soderhall and Cerenius,
1998
). PPOs are produced by haemocytes and released into the
haemolymph (Ashida, 1971
;
Durrant et al., 1993
;
Muller et al., 1999
), from
where they can also be transported to the cuticle through the underlying
cuticular epithelium to facilitate defence against microbial invasion or
abrasion of the cuticle (Asano and Ashida,
2001
; Ashida and Brey,
1995
). There are nine PPO-encoding genes (PPO19)
in A. gambiae (Christophides et
al., 2002
) that show overlapping developmental expression profiles
(Jiang et al., 1997
;
Lee et al., 1998
;
Muller et al., 1999
).
PPO5 and PPO6 are mainly expressed in adult mosquitoes,
whereas PPO14 are expressed in pre-adult stages. Some genes
such as PPO2, PPO3 and PPO9 are induced following blood
feeding (H. M. Muller, unpublished data).
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A. gambiae: an emerging model for the study of innate immunity |
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The A. gambiae genome: a comparative analysis with Drosophila
Anopheles is the first insect vector whose genome has been
sequenced (Holt et al., 2002).
A comparative genome analysis of Drosophila and Anopheles,
which are thought to have diverged approximately 250 million years ago, has
revealed that nearly half the genes are 1:1 orthologues
(Zdobnov et al., 2002
).
Analysis of 18 gene families that include some innate immune-related genes
revealed a twofold deficit in orthologous pairs relative to the genomes as a
whole (Christophides et al.,
2002
). The orthologue-poor families included putative pattern
recognition, signal modulation and effector molecules. Interestingly, immunity
gene families, in particular those involved in pattern recognition, signal
modulation and effector mechanisms, showed substantial species-specific
expansions. Genes belonging to immune signalling pathways are highly
conserved, and this is probably attributed to the multiple functions they
serve, many of which involve developmental processes
(Christophides et al., 2002
;
Zdobnov et al., 2002
). The
expansion and diversification of the PRRs may reflect a strong selective
pressure, leading to faster evolution in the face of distinct microbial floras
prevailing in the ecological habitats of the different species. An extreme
example of this expansion/diversification is the FBN-lectin gene family, which
underwent two independent large expansions: one in Anopheles,
resulting in 52 genes, and the second in Drosophila, resulting in 11
genes; in these species, only two orthologous pairs persist!
Gene discovery and functional analysis in A. gambiae
The A. gambiae genome provides unprecedented opportunities for
mosquito research. For example, whole genome expression analysis using
microarrays is now feasible and is expected to provide new insights into
potential functions of the mosquito immune system during its interactions with
various pathogens, including Plasmodium. Microarray technology is a
very powerful tool for gene discovery; as previously mentioned, a
microarray-based study allowed the identification of numerous genes induced by
both bacterial and Plasmodium challenge
(Dimopoulos et al., 2002);
several of these are currently under in-depth analysis. In the framework of an
international Mosquito Microarray Consortium (MMC) engaging a number of
mosquito groups, our laboratory has constructed a 20 000 EST microarray
(MMC1), and full-genome microarrays based on unique genomic amplicons have
been designed (G. K. Christophides, unpublished data).
Direct and heritable reverse genetics
Gene discovery and generation of hypotheses based on expression patterns
must be complemented with other tools that permit direct assessment of gene
function. For this purpose, a direct reverse genetics method based on
injection of dsRNA into adult mosquitoes has been established
(Blandin et al., 2002), in
addition to using the dsRNA treatment of A. gambiae haemocyte-like
cell lines (Levashina et al.,
2001
). For example, injection of dsRNA corresponding to the
Anopheles defensin gene (DEF1) in the body cavity of adult mosquitoes
efficiently and reproducibly silenced the expression of this gene at the mRNA
and protein levels (Blandin et al.,
2002
). Analysis of the knockout (KO) mosquitoes revealed that
defensin is necessary to combat Gram-positive but not Gram-negative bacterial
infections in vivo; however, the development of P. berghei
was not affected by the absence of defensin. The injected dsRNA is detectable
for at least 12 days post-injection and, hence, is stable enough to mediate a
long-lasting effect. To date, the RNAi approach has been utilized to assay
efficiently the function of several A. gambiae genes in response to
malaria infection, as described below. The RNAi silencing technique was also
established in adult D. melanogaster flies and used to efficiently
silence components of the Toll pathway
(Goto et al., 2003
).
The replacement of wild mosquito populations with genetically modified
strains refractory to Plasmodium development has been widely
postulated as a potential strategy to control malaria transmission. Towards
this aim, substantial effort has been invested to genetically transform
Anopheles species. The early single event of A. gambiae
transformation (Miller et al.,
1987), using the transposable element (TE) P from
Drosophila, proved serendipitous and not transposon mediated. Routine
genetic manipulation methods awaited utilization of TEs that were truly mobile
in mosquitoes and utilization of effective dominant selectable markers. These
tools were developed over the next decade with encouragement from the success
achieved in the medfly, Ceratitis capitata
(Loukeris et al., 1995
), and
the mosquito Ae. aegypti (Coates
et al., 1998
; Jasinskiene et
al., 1998
). The achievement of efficient transformation of the
Asian malaria vector, A. stephensi
(Catteruccia et al., 2000
),
was followed by that of A. gambiae
(Grossman et al., 2001
) and,
more recently, A. albimanus, the south American vector
(Perera et al., 2002
).
With the methods for transgenesis in place, anopheline mosquitoes
refractory to Plasmodium development have been generated.
Bloodmeal-induced expression of two alternative transgenes in A.
stephensi, a short effector peptide
(Ito et al., 2002) and a bee
venom phospholipase (Moreira et al.,
2002
), led to dramatic reduction in oocyst numbers and greatly
impaired transmission of P. berghei to naïve mice. Preliminary
results also suggested that transgenic lines of Ae. aegypti were
rendered resistant to the development of P. gallinaceum through the
bloodmeal-induced, systemic expression of defensin A
(Shin et al., 2003b
).
Although these results illustrate the potential of transgenic technology to
study mosquitoparasite interactions, further refinements are vital to
the development of this field, especially in view of the negative impact of
transformation on mosquito fitness
(Catteruccia et al., 2003).
Clearly, the identification of anopheline promoters that drive transgene
expression in a stage- and tissue-specific manner is a major challenge. These
tools will be essential not only for the expression of effector molecules
targeted against the critical stages of parasite development in the midgut and
salivary gland but also for the silencing of mosquito genes that act as
positive regulators of parasite development. To date, only a few promoters
have been characterized and used to drive gene expression in mosquitoes. These
include Aedes vitellogenin (Shin
et al., 2003a
), Drosophila actin5C
(Brown et al., 2003
) and
Anopheles carboxypeptidase promoter
(Ito et al., 2002
). The
characterization of mosquito promoter sequences could be greatly aided by
utilization of the CreLoxP system, which was shown recently to be
active in Ae. aegypti
(Jasinskiene et al., 2003
).
This system would allow precise functional comparison of alternative promoters
if it is used to target integration of transgenes into the same chromosomal
location: vagaries in expression caused by position effects of the transgene
insertion site would be eliminated, and promoter activity would be easily
compared between transgenic lines.
These approaches will be complemented by the identification of inducible
promoters and conditional expression systems permitting temporal and
tissue-specific control of transgene expression or gene silencing (using
dsRNA-producing transgenes). Such tools will make it possible to finely
monitor effector mechanisms and gene function and to dissect in detail immune
signalling pathways in the vector. Indeed, anopheline strains have been
produced recently that conditionally express transgene in the adult midgut and
haemocytes (G. Lycett and T. G. Loukeris, personal communication). In these
lines, tissue-specific expression is directed by a promoter of a serpin gene,
while conditional regulation is achieved by using the tetracycline
transactivator (Gossen and Bujard,
1992). Transgene expression can be switched on or off by the
supply of tetracycline analogues to the mosquito. With this system,
potentially any stage- or tissue-specific promoter can be made
`inducible'.
Anopheles and Plasmodium: an interplay of immune attack and evasion?
Vector immune responses are believed to account, at least in part, for the
major parasite losses during sporogonic development. In extreme cases, all
Plasmodium parasites are killed in genetically selected refractory
mosquitoes. Anopheles dirus mosquitoes selected for refractoriness
completely block the development of P. yoelii ookinetes by melanotic
encapsulation (Somboon et al.,
1999), and refractory A. gambiae mosquitoes cause the
lysis of P. gallinaceum ookinetes in the cytosol of infected midgut
cells (Vernick et al., 1995
).
The best-studied case of refractoriness is melanotic encapsulation of
Plasmodium ookinetes (Collins et
al., 1986
) in an A. gambiae refractory strain (L35).
Melanization takes place in the extracellular space, between the midgut
epithelial cells and the basal lamina. Genetic mapping of the L35 phenotype
revealed one major (Pen1) and two minor (Pen2 and
Pen3) quantitative trait loci (QTL) implicated in this response
against P. cynomolgi B (Zheng et
al., 1997
). A recent study revealed that refractoriness of L35
mosquitoes to P. cynomolgi Ceylon, a different but related species,
is controlled by at least three QTLs (Pcen2R, 3R and 3L)
(Zheng et al., 2003
).
Interestingly, while Pcen2R and 3L map near Pen3
and Pen2, respectively, and may actually be Pen3 and
Pen2, Pcen3R represents a novel QTL unrelated to Pen1,
suggesting that different genetic loci may be involved in responses to
different malaria parasites. Sequencing of 528 kb of DNA from the
Pen1 region revealed a remarkable number of sequence polymorphisms
that constitute two alternative haplotypes over at least 121 kb
(Thomasova et al., 2002
). The
significance of these haplotypes, and more generally the molecular basis of
the complete melanotic phenotype, is not yet fully understood, although L35
mosquitoes show a high level of reactive oxygen species, which is further
enhanced by bloodfeeding (Kumar et al.,
2003
). Similarly, the molecular basis of P. gallinaceum
lysis in a refractory strain of A. gambiae
(Vernick et al., 1995
) is
still unknown. However, recent studies showed that the melanotic response of
L35 mosquitoes can be reversed by silencing specific A. gambiae
immunity genes (Blandin et al.,
2004
; G. K. Christophides, unpublished data).
Melanization of P. falciparum ookinetes is rare in infected
field-caught mosquitoes (Niare et al.,
2002; Schwartz and Koella,
2002
). Rather, these mosquitoes are characterized by a high
natural frequency of segregating resistance alleles that apparently attenuate
the intensity of infection in the vector
(Niare et al., 2002
). This
suggests that P. falciparum induces a strong selective pressure on
Anopheles. Therefore, the parasite and its vector most likely
represent a co-evolving system in dynamic equilibrium. The fact that the L35
strain of A. gambiae melanizes several species of malaria parasites,
including P. berghei, P. gallinaceum, P. cynomolgi B and allopatric
but not sympatric strains of P. falciparum
(Collins et al., 1986
), adds
further support to this hypothesis. Several questions arise concerning
mosquito factors that may specifically protect sympatric ookinetes and the
means by which the parasite may evade or subvert the mosquito immune
responses. A recent high-throughput proteomic approach has revealed that
P. falciparum sporozoites express several proteins of the
var gene family and other surface receptors
(Florens et al., 2002
) that
were initially thought to be restricted to the mature asexual stages of the
parasite. The fact that var genes are involved in immune evasion in
the vertebrate host makes it tempting to explore whether var or other
specific parasite surface proteins can mediate immune evasion in the vector as
well as the vertebrate host.
Protozoan pathogens have evolved several mechanisms to evade the immune
responses of the vertebrate host, including antigenic variation, shedding of
surface proteins, antigenic mimicry, hiding inside cells and modulation of the
host immune responses (Zambrano-Villa et
al., 2002). Until recently, the molecular mechanisms that control
the number of these parasites in their mosquito vectors, thus facilitating
their transmission to vertebrates, remained uncharacterized. They could only
be proposed by analogy to antibacterial immunity, by in vitro studies
or by inference from descriptions of gene expression patterns. However,
application of the dsRNA-mediated gene silencing technique in vivo
has changed this situation radically. Recently, specific mosquito gene
products, which act as antagonists of parasite development, have been
identified in living mosquitoes, as well as others that act as agonists
protecting the parasite against antagonists. The antagonists identified to
date are the thioester-containing protein TEP1 and a leucine-rich protein,
LRIM1. TEP1 is an acute-phase haemocyte-specific protein previously shown to
bind and opsonize bacteria in a thioester-dependent manner
(Levashina et al., 2001
).
Recently, TEP1 was also shown to be involved in the killing of P.
berghei ookinetes as they cross the midgut epithelium of A.
gambiae (Blandin et al.,
2004
). This was supported by several lines of evidence: (1) TEP1
knockout induces a fivefold increase in ookinete survival in a suceptible (G3)
strain of A. gambiae and permits the successful development of P.
berghei in a refractory (L35) strain of the same species; (2) TEP1 binds
to the surface of ookinetes after they cross the midgut epithelium in both
strains, but the timing of binding differs between these strains, and (3)
TEP1-associated ookinetes display degeneration, evidenced by parasite
blebbing, loss of the vital fluorescent marker GFP, nuclear abnormalities and
fragmentation, and perturbations in the distribution of the ookinete-specific
surface protein P28. Interestingly, 100% of ookinetes are killed in the L35
strain as compared with 80% in the G3 strain. The identification of TEP1
staining on live, morphologically normal ookinetes has suggested that TEP1
first binds to the ookinetes and then leads to their degeneration. Two
polymorphic alleles of TEP1 were identified: TEP1r is
associated with the L35 strain and TEP1s with the susceptible strain.
However, conclusive evidence is still missing as to whether these polymorphic
alleles are associated with the faster binding of TEP1 and more efficient
killing of ookinetes in the L35 strain.
In parallel studies (Osta et al.,
2004), LRIM1 was identified as a new parasite antagonist whose
absence induces a dramatic, nearly fourfold, increase in the number of
parasites in susceptible mosquitoes. LRIM1 is also predominantly expressed in
the carcass (mosquito remnant following midgut isolation) as compared with the
midgut and is specifically upregulated in the carcass of infected mosquitoes
as compared with non-infected mosquitoes. Interestingly, LRIM1 expression in
the midgut is strong and transient: upregulation occurs at 2428 h
post-infection, coinciding with the period of ookinete invasion of the midgut
epithelium. Additional experiments revealed that two A. gambiae
C-type lectins, CTL4 and CTLMA2, act as agonists protecting the parasite from
mosquito immune responses (Osta et al.,
2004
). Silencing either lectin gene by RNAi induces massive
melanization of ookinetes in the susceptible A. gambiae G3 strain:
the CTL4 KO results in melanization of nearly all ookinetes, while partial
melanization is observed in CTLMA2 KO mosquitoes. Both lectins are
predominantly expressed in the mosquito carcass (possibly in fat body and/or
haemocytes) as compared with the midgut and are specifically upregulated in
the carcass during ookinete invasion of the midgut epithelium, suggesting some
immune signalling between tissues. These results highlight the potential use
of CTL genes or proteins as targets to block Plasmodium transmission
in the vector.
Genetic epistasis analysis revealed that the melanization response induced in the absence of CTLs requires LRIM1 function: the double KO of LRIM1 with either lectin gene completely abolishes the melanization phenotype and induces a fourfold increase in oocyst numbers, a phenotype similar to that of the single LRIM1 KO. Further research is being conducted to address the detailed mechanisms by which LRIM1 dramatically limits the parasite load in the vector, while CTLs protect the parasite. For now, our understanding can be summarized as in Fig. 3.
|
It is clear that parasite transmission depends upon complex molecular and
cellular interactions acting at different levels of the parasite's life cycle
in the vector (Alavi et al.,
2003). Deciphering these interactions and identifying the
molecules (defence or non-defence) that negatively and positively regulate
parasite development in the vector will provide valuable information that can
be exploited in designing novel transmission-blocking strategies for
vector-borne pathogens. It will obviously be a matter of considerable
importance to determine whether TEP1, LRIM1, CTL4 and CTLMA2 are induced by
and act on P. falciparum in the same manner as on P.
berghei.
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Concluding remarks and perspectives |
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List of abbreviations |
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Acknowledgments |
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![]() |
Footnotes |
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References |
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