Blocking malaria parasite invasion of mosquito salivary glands
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA
(e-mail: aajames{at}uci.edu)
Accepted 21 July 2003
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Summary |
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Key words: sporozoite, salivary gland, transgenic, mosquito, genetic control, lectin, blocking antibody
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Introduction |
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Mosquito salivary glands |
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In general, the three lobes of male salivary glands appear similar to one
another and likely all have the same secretory capabilities
(James, 1994). Female glands
are differentiated into two lateral and one medial lobes. The proximal regions
of the lateral lobes in females express and secrete salivary gland products
such as amylases and
1-4 glucosidase that are involved in sugar
feeding, and these lobes appear to overlap functionally the male salivary
glands (James, 1994
;
Arcá et al., 1999
). In
contrast, the medial lobe and distal-lateral lobes express genes whose
products such as apyrases, anticoagulants and vasodilatory agents are involved
in hematophagy (Champagne et al.,
1995
; Smartt et al.,
1995
; Beerntsen et al.,
1999
; Stark and James,
1998
; Arcá et al.,
1999
).
In addition to their gene expression characteristics, the surface
properties of the different salivary gland lobes are also variable. A number
of studies with lectins and monoclonal antibodies raised to whole salivary
glands show differential binding of these agents to the lobes of the female
glands (Perrone et al., 1986;
Barreau et al., 1995
,
1999
). Some of these reagents
recognize specifically the distal-lateral and/or medial lobes, indicating a
differentiation among the regions of the glands. These differences are
particularly important because the results of a number of studies have been
interpreted to indicate that sporozoites preferentially invade the
distal-lateral and medial lobes of the female glands
(Sterling et al., 1973
;
Rossignol et al., 1984
;
Golenda et al., 1990
;
Pimenta et al., 1994
). In a
striking demonstration of this specificity, a peptide, SM1, binds to the
distal-lateral and medial lobes of female glands of An. gambiae and
An. stephensi, and blocks slightly more than 90% of P.
berghei sporozoite invasion in the latter species
(Ghosh et al., 2001
). The
authors conclude that the peptide competes with the sporozoites for a salivary
ligand.
Some of the most exciting work being done in vector physiology is the
discovery and characterization of a large number of proteins and their
corresponding genes that are involved in facilitating hematophagy. Classes of
proteins that appear common to all bloodfeeding arthropods include
polyphyletic groups of enzymes that prevent coagulation, cause vasodilation
and prevent platelet aggregation (Stark
and James, 1996). Furthermore, proteomics approaches have provided
comprehensive lists of the individual gene products in the `sialomes' of
various mosquito vectors including Aedes aegypti, Anopheles gambiae
and An. stephensi (Valenzuela et al.,
2002
,
2003
;
Francischetti et al., 2002
).
These studies have revealed an amazing diversity in the recruitment of members
of gene families to roles in hematophagy as well as a remarkable amount of
apparent redundancy in each recognized functional class. For example, proteins
that function as vasodilatory agents include small peptides, the sialokinins,
from Ae. aegypti (Champagne et
al., 1995
), and larger enzymes, the catechol oxidases/peroxidases,
in An. albimanus (Ribeiro and
Nussenzveig, 1993
). Furthermore, the products of a number of
different genes function as anti-coagulants in individual mosquito species
(Stark and James, 1998
;
Valenzuela et al., 2002
,
2003
;
Francischetti et al.,
2002
).
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Parasite-salivary gland interactions |
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The invasion of salivary glands by sporozoites is thought to be mediated by
receptor-ligand-like interactions resulting from the binding of parasite
surface ligands to specific receptors on the salivary glands
(Beerntsen et al., 2000). The
receptor-ligand hypothesis derives support from observations that the majority
of sporozoites released from the oocysts are found in the salivary glands
(Golenda et al., 1990
). This
is interpreted to indicate that sporozoites have some mechanism for
differentiating among the multiple mosquito organs suspended in the hemocoel.
Electron microscope studies of sporozoite interactions with salivary glands
also lend support for a receptor-ligand model. Analyses of P.
gallinaceum invasion of Ae. aegypti glands show filamentous
attachments of the sporozoites to the basal lamina of the glands, suggesting
the presence of a receptor-ligand complex
(Pimenta et al., 1994
).
Interestingly, initial contact of the sporozoite with the gland was followed
by a reorientation of the sporozoite so that the `anterior tip' (apical end)
is in close association with the plasma membrane of the salivary gland cells.
This behavior strongly supports the hypothesis that additional parasite
surface molecules are needed for invasion of the gland following the initial
binding.
Rosenberg (1985) showed
that there were species-specific recognition properties of sporozoites for
salivary glands. Plasmodium knowlesi sporozoites could recognize and
invade salivary glands from An. dirus even when the glands were
transplanted to a non-permissive host, An. freeborni. Conversely,
these sporozoites could not infect An. freeborni salivary glands
under any circumstances. These experiments have been interpreted by many to
indicate that the presence (An. dirus) or absence (An.
freeborni) of species-specific receptor molecules is an important
component of vector competence for sporozoites to invade the salivary
glands.
Potential parasite ligands for salivary gland recognition include the
circumsporozoite protein (CSP). The CSP is the major protein on the surface of
sporozoites, and may account for as much as 10% of the protein located there
(Nussenzweig and Nussenzweig, 1989). Sidjanski et al.
(1997) bound recombinant
P. falciparum CSP to the medial (strongly) and distal-lateral
(weakly) lobes of An. stephensi salivary glands. This group was able
to show that a specific domain (region 1) was the likely salivary binding
domain of CSP. Others have shown that CSP shed during invasion of glands by
sporozoites remains on the surface of the glands
(Posthuma et al., 1988
;
Golenda et al., 1990
).
Furthermore, some of the monoclonal antibodies made to P. gallinaceum
CSP blocked sporozoite invasion of Ae. aegypti salivary glands
(Warburg et al., 1992
). These
results could occur as a consequence of blocking a receptor or steric
hindrance. Interestingly, the SM1 peptide is not similar in amino acid
sequence to the CSP (Ghosh et al.,
2001
), indicating that other molecules also could function as
ligands.
Other molecules on the surface of sporozoites such as the
thrombospondin-related anonymous protein (TRAP) and the apical membrane
antigen/erythrocyte binding-like protein (MAEBL) may have more complex
interactions with the salivary glands (reviewed in
Kappe et al., 2003). These
proteins may be involved with the invasion phase of sporozoite infection that
occurs after the initial attachment.
Surprisingly, the putative salivary gland receptor molecules have yet to be identified. Despite the long-time availability of reagents (lectins, monoclonal antibodies and peptides) that compete or block sporozoite invasion of the glands, the molecules with which these reagents interact remain elusive. The molecular complexity of the basement membrane surrounding the salivary gland epithelium may make difficult the identification of a single molecule that functions as the receptor. Furthermore, there may not be a single molecule that fulfills this role, but multiple interchangeable molecules or a molecular complex may be the sporozoite target.
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Antisporozoite refractory phenotypes |
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Sporozoites are potentially vulnerable when they are in the salivary glands. Promoters from genes expressed specifically in the glands can be used to drive the expression of an effector molecule that would disable the parasites and prevent them from being secreted or abrogate their infectivity to the vertebrate host.
The most attractive target is provided by the sporozoites as they make
their way through the hemolymph from the oocysts to the salivary glands. This
approach provides the opportunity to bathe the sporozoite in a solution
(hemolymph) containing an effector molecule. Furthermore, the mosquito has a
number of genes that express proteins such as vitellogeninins and lipophorins
that are made in the fat body and then transported into the hemolymph
(Raikhel et al., 2002;
van Heusden et al., 1998
). The
promoters and other control sequences of these genes direct the expression of
large amounts of the corresponding proteins and therefore these sequences are
good candidates for donating the control portions of synthetic effector
genes.
Various effector strategies and molecules were reviewed recently by Nirmala
and James (2003). They
described five categories of approaches, based on the target of action.
Effector molecules can interact with ligands on the parasite surface, or their
corresponding receptors on mosquito tissues. They can block the activity of
parasite-expressed proteins important for invasion of tissues, or they can be
toxins that destroy parasites. Finally, immune-system components could be
regulated and expressed in the vector to incapacitate the parasite. A list of
approaches that have been applied to salivary glands is presented in
Table 1.
|
Effector molecules targeting salivary gland receptors were some of the
first to be tested for transmission blocking. Carbohydrate moieties were
implicated in parasite receptor recognition when it was shown that P.
gallinaceum sporozoites could not invade salivary glands of Aedes
aegypti treated with lectins (Barreau
et al., 1995). Plasmodium berghei numbers are reduced in
mosquitoes treated with a peptide, SM1, which binds to An. stephensi
midguts and salivary glands (Ito et al.,
2002
). Transgenic mosquitoes expressing SM1 have fewer oocysts,
and concomitantly fewer sporozoites, when compared with controls. It is
intriguing that SM1 binds both the apical surface of the midgut and the basal
surface of the distal lobes of the salivary glands, suggesting the presence of
similar receptors on these organs.
Single-chain antibody fragments (scFv) composed of fused heavy- and
light-chain variable regions can preserve the specificity of an antibody and
be expressed as the product of a single gene. An scFv that binds CSP, N2scFv,
reduced by 99% the number of P. gallinaceum sporozoites in salivary
glands (Capurro et al., 2000).
These studies demonstrate that parasite ligands are good targets for effector
molecules. Furthermore, it is anticipated that targeting the parasite will
impose less of a genetic load on the transgenic mosquito than will interfering
with surface molecules on mosquito tissues.
Immune system interactions with sporozoites could provide the basis of an
effector strategy. Both exogenously and endogenously derived immune peptides
have been evaluated for their effects on malaria parasites. Defensins isolated
from Aeschna cyanea and Phormia terranovae reduced the
number of viable P. gallinaceum oocysts and sporozoites by 50% when
injected into Ae. aegypti
(Shahabuddin et al., 1998).
Natural immune responses of mosquitoes to infection are also being studied as
possible effector mechanisms for transmission blocking. Microarray analyses
coupled with the sequence of the An. gambiae genome have provided the
first comprehensive look at immune responses in parasite-infected mosquitoes
(Dimopoulos et al., 2003; Holt et al.,
2002
), and there are possibilities of modulating responses that
will affect sporozoite development.
The development of antisporozoite effector genes is ongoing work. Combining these genes with others that target ookinetes and prevent oocyst formation should permit producing a multigenic phenotype of `no sporozoites' in the salivary glands. Furthermore, the use of multiple effector genes maybe necessary to prevent the selection of resistance to any one mechanism. This could prevent the breakdown of a control strategy based on a genetics approach. In addition, a balance among fitness effects, effectiveness of the molecule, ease of engineering of the phenotypes and mechanism for spreading the phenotypes through a population will dictate the practicality of any one strategy. Ultimately, saving lives will be the most important measure of these approaches.
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Acknowledgments |
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References |
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