Laminin and the malaria parasite's journey through the mosquito midgut
1 Institute of Molecular Biology and Biotechnology, Foundation for Research
and Technology Hellas, Vassilika Vouton, PO Box 1527, 71110 Heraklion,
Crete, Greece
2 European Molecular Biology Laboratory, Meyerhofstr. 1, 69000 Heidelberg,
Germany
3 Department of Biology, University of Crete, 71110 Heraklion, Crete,
Greece
Author for correspondence (e-mail:
louis{at}imbb.forth.gr)
Accepted 19 April 2005
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Summary |
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Key words: Anopheles gambiae, basal lamina, ookinete, Plasmodium berghei, RNAi
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Introduction |
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In the context of vectorparasite interactions, the possibility that
the Anopheles midgut basal lamina could be involved in additional
functions other than the obvious one, namely that of a physical barrier that
prevents ookinetes from continuing their passage, has often been considered.
It has been known for several decades that parasites injected into the
haemocoel can develop further upon binding onto the mosquito midgut wall
(Weathersby, 1952;
Warburg and Schneider, 1993
),
and this was confirmed in a dramatic way when it was shown that the injection
of parasites into Drosophila melanogaster also leads to the
development of infective sporozoites within oocysts attached to the basal
lamina at multiple sites, not just the midgut
(Schneider and Shahabuddin,
2000
). Thus, binding of the ookinete to the basal lamina is a
condition that supports its development into a functional oocyst, as
demonstrated with the critical requirement of Matrigel in the successful
culture of Plasmodium berghei in vitro
(Al-Olayan et al., 2002
).
Although laminin is only one of many constituents of the basal lamina, the
fact that it has been shown to interact directly with invading parasites
suggests a crucial role in the invasion process. To investigate this
possibility, we temporally followed, using confocal microscopy and
immunohistochemical techniques, the localisation of laminin during different
stages of P. berghei parasite development in the Anopheles
gambiae midgut. Moreover, to observe whether the biochemical interactions
can indeed have a functional role, we used the recently described RNAi
technique (Fire et al., 1998)
to determine whether a reduction in the transcription of mosquito laminin
1 would lead to a novel infection phenotype. Our results indicate that
laminin does indeed interact with the parasite and plays a crucial role in the
parasite's development within the mosquito midgut.
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Materials and methods |
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Double-stranded RNA production and injection
Double-stranded (ds) RNA was produced as described previously using the
plasmid pLL6ds for control dsGFP (ds green fluorescent protein;
Levashina et al., 2001) and
pLLlam for laminin (dsLANB2). In order to construct pLLlam, a 750-bp fragment
(covering the 3' UTR) was PCR-amplified from pLAM-BamHI, which
contains almost the entire LANB2 gene that encodes laminin
1
(Vlachou et al., 2001
). The
primers used were FPNotL (TTG CGG CCG CAA GCA GCA GCA CTA GCA GTA GTA) and
RPBamL (CGG ATC CGG TTA TCT TCT GCG GCA CG). The fragment was cloned between
the two T7 promoters of the plasmid pLL6, to create pLLlam. Sense and
antisense RNAs were synthesized using the T7 Ambion (Austin, TX, USA)
Megascript kit, annealed in water, and stored as dsRNA at 80°C
until use. A nanoinjector (Nanoject; Drummond, Burton, OH, USA) was used to
inject 69 nl of dsRNA (2 µg µl-1) into the thorax of
CO2-anaesthetized 12-day-old female mosquitoes as described
by Blandin et al. (2002
). The
mosquitoes were allowed to recover for 4 days before being fed on infected
mice.
RNA analysis with quantitative real-time PCR
Total RNA was extracted from 10 injected mosquito guts using TRIzol reagent
(Gibco BRL, Gaithersburg, MD, USA) 4 days post-injection, and 24 h after blood
feeding. First-strand cDNA was synthesized using Superscript II Reverse
Transcriptase (Invitrogen, Carlsbad, CA, USA). Plasmids to be used as
standards for qPCR were constructed by inserting gene fragments from the
coding regions of either the LANB2 or S7 gene into the pGEM
T-easy vector (Promega, Madison, WI, USA). The concentration of the plasmids
used as standards ranged from 1 ng to 10 fg to determine copy numbers of the
target inserts. Laminin-specific primers were selected that do not overlap
with the region of the LANB2 gene used to construct the dsRNA:
FPLAM2, 5'-GCTAAGACGGACAACCGACTG-3'; RPLAM2,
5'-TCTCGGCAGCACTCAGACG-3'. S7 primers (forward,
5'-GTGCGCGAGTTGGAGAAGA-3'; reverse,
5'-ATCGGTTTGGGCAGAATGC-3') were used as internal standards
(Salazar et al., 1993). Using
the QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA, USA), PCR reactions
were carried out in a continuous fluorescence detector gradient cycler
(OPTICON; MJ Research, Waltham, MA, USA) over 35 cycles of 95°C for 15 s,
60°C (LANB2 235 bp product) or 55°C (S7
76 bp product) for 30 s and 72°C for 30 s, followed by one cycle
of continuous monitoring of fluorescence from 60°C to 94°C to generate
the melting curve of the PCR products. Different primers and conditions were
used for semi-quantitative PCR: FPLAM, 5'-CAAACAGCCCAGGACAAGTA-3';
RPLAM, 5'-TTACGGTTCCAGATCGT-3'. The primers produce a 551-bp
fragment using a standard PCR program (30 s at 95°C; 45 s at 56.5°C;
45 s at 72°C) for 29 cycles. The S7 standard
(Salazar et al., 1993
) was
amplified with 19 cycles using the same conditions. Each experiment was
repeated three times.
Plasmodium infections
Within each experiment, dsGFP- and dsLANB2-injected mosquitoes (4 days
after the injection) were simultaneously fed on the same mouse that was
infected with a GFP-expressing Plasmodium berghei clone
(Vlachou et al., 2004).
Mosquito guts were dissected 7 days post-blood meal and fixed. Oocysts were
counted using a UV-light fluorescent microscope. Thirty mosquitoes were
injected per treatment (i.e. GFP or LAM), and each series of injections was
repeated four times.
Statistical analysis
Analysis was carried out using the MINITAB 13 statistical package. Since
none of the data were normally distributed, they were analysed using the
MannWhitney U-test for non-parametric data.
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Results |
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When the ookinetes reach the basal lamina, they transform into the rounded oocysts that will subsequently initiate sporogony. Fig. 1C shows one young oocyst, 3 days after the ingestion of the blood meal, counterstained with the green fluorescing anti-P28 antibody. Under higher magnification (two insets), laminin is observed coating the parasite. At day 10 (Fig. 1D), a thin line of anti-laminin antibody is still seen to label the oocyst surface, although it is difficult to determine whether there is an intimate contact between the surface of the parasite and the extracellular matrix. Finally, 15 days after the ingestion of the blood meal, the by now much larger oocysts are covered by a thin layer of laminin, but this may not be bound to the oocyst as such. Probably, this represents the stretching out of the basal lamina as the oocyst grows in size. A representative oocyst at this stage is shown protruding from the muscles in Fig. 1D.
The intimate association of mosquito laminin and the parasites ends upon rupture of the oocyst approximately 15 days after the blood meal. In a low magnification of the midgut (Fig. 1F), green fluorescing sporozoites can be seen on the haemocoel-side of the midgut musculature, while a very late-stage oocyst can be seen at the right-hand side, defined by the strong CSP staining (blue: nuclei of still immature oocysts and epithelial nuclei). Lastly, in Fig. 1G, the sporozoites are clearly seen on the muscles, while a circular black area within a rectangle of laminin-covered muscles represents an immature (non CSP-expressing) oocyst.
Invasion and oocyst formation are influenced by expression of laminin 1 mRNA
In a previous report (Vlachou et al.,
2001), we had determined that one of the mosquito laminins,
laminin
1, could specifically bind the Plasmodium surface
proteins P25 and P28. Moreover, it was later shown that the same laminin could
also bind to the SOAP (Dessens et al.,
2003
) and CTRP (Mahairaki et
al., 2005
) proteins of the ookinete. In order to investigate
whether these biochemical lamininparasite interactions and the
association described above also serve a biological role in the survival of
the parasite during the invasion of the mosquito midgut by P. berghei
parasites, we took advantage of the dsRNA gene silencing technology
(Fire et al., 1998
). This
method has previously been used to assess gene function both in
Anopheles cells (Levashina et
al., 2001
) and in whole mosquitoes (Blandin et al.,
2002
,
2004
;
Osta et al., 2004
).
Double-stranded RNA corresponding to the gene to be silenced is produced
in vitro and injected into adult mosquitoes. The mosquitoes are then
fed on infected mice, and oocyst numbers are counted after one week. The
construct that was used to silence LANB2 contained a 756 bp fragment
derived from the 3' UTR region of the laminin
1 transcript
(nucleotides 52696025; see Fig.
2A). BLAST analysis determined that this is a single-copy segment
of the genome of An. gambiae (not shown). As a control, dsRNA
transcribed in vitro from a construct containing the gene for GFP
(green fluorescent protein) was injected in parallel.
|
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We then proceeded to test the effect of knocking down the laminin 1
expression on parasite development. Double-stranded RNA was injected as
described above and, after recovery for 4 days, the injected mosquitoes (both
experimental and control) were fed on the same P. berghei-infected
mice. Several mosquitoes from each experiment did not survive the injections,
leading to a reduction in the total number of midguts analysed. It is
important to note, though, that there was no significant difference in the
survival of the mosquitoes injected with the GFP dsRNA (85/120) or the laminin
1 (79/120). Midguts were dissected 7 days later and the number of
oocysts was counted under the microscope. The results of these experiments are
shown in Fig. 2B. The mean
number of oocysts observed in 85 mosquitoes injected with GFP dsRNA was 42.5
per midgut. By contrast, in 79 females injected with laminin
1 dsRNA,
the mean number was 17.5 per midgut, a significant reduction in oocyst levels
(P<0.0001). Taken together, this indicates that there was an
approximately 60% reduction in oocyst formation when LANB2 expression
was suppressed.
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Discussion |
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The fact that laminin is an integral component of the An. gambiae
basal lamina (Vlachou et al.,
2001), a structure that is reached by ookinetes only after the
primary invasion of, and exit from, an epithelial cell, implies that its role
is different from that of a (putative) receptor that would be mediating entry
into the epithelium. However, the idea of laminin acting as a receptor to
`trigger' ookinete transformation into an oocyst has been suggested by Arrighi
and Hurd (2002
). Furthermore,
the fact that laminin is found coating the young oocysts at day 3 of
development, and is seen in close vicinity to older oocysts 10 days after the
uptake of the infected blood meal, suggests an ongoing role, at least for the
first half of sporogony. Previous studies have also shown a close association
between both ookinetes and oocysts with the basal lamina during sporogonic
development (Meis et al.,
1989
,
1992
). One potential function
of the interaction (or association) that fits these time constraints would be
a masking of the developing parasite from possible attacks by the insect's
immune system. Studies by Rizki and Rizki
(1984
) in Drosophila
suggest that foreign bodies lacking a `coating' of basal lamina are attacked
and melanized. In addition, Paskewitz and Riehle
(1998
) demonstrated that
pre-incubation of Sephadex beads in a susceptible strain of An.
gambiae led to a significant reduction in melanization when the beads
were transferred to a refractory strain. Obviously, our results cannot provide
a definite answer on the `masking theory', yet this remains an attractive
hypothesis.
The effect of knocking-down the LANB2 gene on parasite development
indicates that the basal lamina and its integrity are vital for parasite
development. It is likely that the effects observed are due to an overall
reduction of fully functional basal lamina and not that the decrease of
laminin 1 per se has a direct effect on the development of
P. berghei, although this latter possibility cannot be excluded.
Unfortunately, it is impossible to use the antibody stains to make either a
quantitative or a qualitative statement in this respect.
The basal lamina is a structure that is formed early in development during
the pupal stages when the imaginal structures are differentiated
(Clements, 1992). Since the
midgut is formed quasi de novo, the majority of the surrounding basal
lamina is also synthesized during the same period. Thus, a reduction in
expression of the gene was not necessarily expected in the adult stage. On the
other hand, the blood meal is accompanied by an extreme extension of the
midgut, a concomitant change in the epithelium to become squamous
(Billingsley, 1990
) and, as we
have shown here, an increase in midgut-specific laminin expression. It is
obvious that this extension includes the surrounding basal lamina, as a
previous report by Reinhardt and Hecker
(1973
) showed that the basal
lamina is thicker following digestion of a blood meal. Therefore, a new
synthesis of some or all of its components could be considered to be normal in
order to facilitate the reorganisation of cellular morphology and to repair
possible physical damage. Such a model for basal lamina protein expression
during parasite development has been proposed by Gare et al.
(2003
). This phenomenon of
continuous production and reorganization has also been described in vertebrate
extracellular matrices (Schwarzbauer,
1999
). In insects, it has been suggested that a specific type of
hemocytes may be able to synthesize and secrete basal lamina components
(Fessler and Fessler, 1989
).
In fact, it may be worth considering the possibility that the binding of
laminin to the surface of ookinetes and oocysts may be due to an indirect
effect of basal lamina protein processing. Perhaps Plasmodium
parasites exploit this synthesis of laminin in order to achieve a more
efficient infection of the insect host.
This report adds further evidence to the list of observations that suggest
that laminin is a component that is indeed involved in the invasion and
development of the malaria parasite within the mosquito midgut, thereby
supporting the notion that components of the basal lamina play an important
role in the malaria parasite's sporogonic cycle. This was suggested earlier in
two in vitro systems (Warburg and
Schneider, 1993; Al-Olayan et
al., 2002
), although it was not necessary in others
(Siden-Kiamos et al., 2000
).
Nevertheless, it remains to be seen what the exact role of the basal lamina is
in this process.
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Acknowledgments |
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Footnotes |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adini, A. and Warburg, A. (1999). Interaction of Plasmodium gallinaceum ookinetes and oocysts with extracellular matrix proteins. Parasitology 119,331 -336.[CrossRef][Medline]
Al-Olayan, E. M., Beetsma, A. L., Butcher, G. A., Sinden, R. E.
and Hurd, H. (2002). Complete development of mosquito phases
of the malaria parasite in vitro. Science
295,677
-679.
Arrighi, R. B. G. and Hurd, H. (2002). The role of Plasmodium berghei ookinete proteins in binding to basal lamina components and transformation into oocysts. Int. J. Parasitol. 32,91 -98.[CrossRef][Medline]
Billingsley, P. F. T. (1990). The midgut ultrastructure of hematophagous insects. Annu. Rev. Entomol. 35,219 -248.[CrossRef]
Blandin, S., Moita, L. F., Kocher, T., Wilm, M., Kafatos, F. C.
and Levashina, E. A. (2002). Reverse genetics in the mosquito
Anopheles gambiae: targeted disruption of the Defensin gene.
EMBO Rep. 3,852
-856.
Blandin, S., Shiao, S. H., Moita, L. F., Janse, C. J., Waters, A. P., Kafatos, F. C. and Levashina, E. A. (2004). Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell. 116,661 -670.[CrossRef][Medline]
Clements, A. N. (1992). Metamorphosis. In The Biology Of Mosquitoes, vol.1 , pp. 171-193. New York: Chapman and Hall.
Dessens, J. T., Beetsma, A., Dimopoulos, G., Wengelnik, K.,
Crisanti, A., Kafatos, F. C. and Sinden, R. E. (1999). CTRP
is essential for mosquito infection by malaria ookinetes. EMBO
J. 18,6221
-6227.
Dessens, J. T., Siden-Kiamos, I., Mendoza, J., Mahairaki, V., Khater, E., Vlachou, D., Xu, X., Kafatos, F. C., Louis, C., Dimopoulos, G. et al. (2003). SOAP, a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development. Mol. Microbiol. 49,319 -329.[CrossRef][Medline]
Fessler, J. H. and Fessler, L. I. (1989). Drosophila extracellular matrix. Annu. Rev. Cell. Biol. 5,309 -339.[CrossRef][Medline]
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature 391,806 -811.[CrossRef][Medline]
Gare, D. C., Piertney, S. B. and Billinglsey, P. F. (2003). Anopheles gambiae collagen IV genes: cloning, phylogeny, and midgut expression associated with blood feeding and Plasmodium infection. Int. J. Parasitol. 33,681 -690.[CrossRef][Medline]
Hynes, R. O. and Zhao, Q. (2000). The evolution of cell adhesion. J. Cell Biol. 150,F89 -F95.[CrossRef][Medline]
Kappe, S. H., Kaiser, K. and Matuschewski, K. (2003). The Plasmodium sporozoite journey: a rite of passage. Trends Parasitol. 19,135 -143.[CrossRef][Medline]
Levashina, E. A., Moita, L. F., Blandin, S., Vriend, G., Lageux, M. and Kafatos, F. C. (2001). Conserved role of a component-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae. Cell 104,709 -718.[CrossRef][Medline]
Mahairaki, V., Voyatzi, T., Siden-Kiamos, I. and Louis, C. (2005). The Anopheles gambiae gamma1 laminin directly binds the Plasmodium berghei circumsporozoite- and TRAP-related protein (CTRP). Mol. Biochem. Parasitol. 140,119 -121.[CrossRef][Medline]
Meis, J. F., Pool, G., van Gemert, G. J., Lensen, A. H., Ponnudurai, T. and Meuwissen, J. H. (1989). Plasmodium falciparum ookinetes migrate intercellularly through Anopheles stephensi midgut epithelium. Parasitol. Res. 76, 1,13 -19.[CrossRef][Medline]
Meis, J. F., Wismans, P. G., Jap, P. H., Lensen, A. H. and Ponnudurai, T. (1992). A scanning electron microscopic study of the sporogonic development of Plasmodium falciparum in Anopheles stephensi. Acta Trop. 50,227 -236.[CrossRef][Medline]
Miosge, N. (2001). The ultrastructural composition of basement membranes in vivo. Histol. Histopathol. 16,1239 -1248.[Medline]
Osta, M. A., Christophides, G. K. and Kafatos, F. C.
(2004). Effects of mosquito genes on Plasmodium
development. Science
303,2030
-2032.
Paskewitz, S. M. and Riehle, M. (1998). A factor preventing melanization of Sephadex CM-25 beads in Plasmodium-susceptible and refractory Anopheles gambiae.Exp. Parasitol. 90,34 -41.[CrossRef][Medline]
Reinhardt, C. and Hecker, H. (1973). Structure and function of the basal lamina and of the cell junctions in the midgut epithelium (stomach) of female Aedes aegypti L. (Insecta, Diptera). Acta Trop. 30, 4,213 -236.[Medline]
Rizki, T. and Rizki, R. M. (1984). The cellular defense system of Drosophila melanogaster. Insect Ultrastruct. 2,579 -603.
Salazar, C. E., Mills-Hamm, D., Kumar, V. and Collins, F. H. (1993). Sequence of a cDNA from the mosquito Anopheles gambiae encoding a homologue of human ribosomal protein S7. Nucleic Acids Res. 21,4147 .[Medline]
Schneider, D. and Shahabuddin, M. (2000).
Malaria parasite Development in a Drosophila model.
Science 288,2376
-2379.
Schwarzbauer, J. (1999). Basement membrane: Putting up the barriers. Curr. Biol. 9,R242 -R244.[CrossRef][Medline]
Siden-Kiamos, I. and Louis, C. (2004). Interactions between malaria parasites and their mosquito hosts in the midgut. Insect Biochem. Mol. Biol. 34,679 -685.[CrossRef][Medline]
Siden-Kiamos, I., Vlachou, D., Margos, G., Beetsma, A., Waters,
A. P., Sinden, R. E. and Louis, C. (2000). Distinct roles for
Pbs21 and Pbs25 in the in vitro ookinete to oocyst transformation of
Plasmodium berghei. J. Cell Sci.
113,3419
-3426.
Sinden, R. E. (2002). Molecular Interactions between Plasmodium and its insect vectors. Cell. Microbiol. 4,713 -724.[CrossRef][Medline]
Vlachou, D., Lycett, G., Sidén-Kiamos, I., Blass, C., Sinden, R. E. and Louis, C. (2001). Anopheles gambiae laminin interacts with the P25 surface protein of Plasmodium berghei ookinetes. Mol. Biochem. Parasitol. 112,229 -237.[CrossRef][Medline]
Vlachou, D., Zimmermann, T., Cantera, R., Janse, C. J., Waters, A. P. and Kafatos, F. C. (2004). Real-time, in vivo analysis of malaria ookinete locomotion and mosquito midgut invasion. Cell Microbiol. 6,671 -685.[CrossRef][Medline]
Warburg, A. and Schneider, I. (1993). In vitro culture of the mosquito stages of Plasmodium falciparum.Exp. Parasitol. 76,121 -126.[CrossRef][Medline]
Weathersby, A. B. (1952). The role of the stomach wall in the exogenous development of Plasmodium gallinaceum as studied by means of haemocoel injections of susceptible and refractory mosquitoes. J. Infect. Dis. 91,198 -205.[Medline]