A snake venom phospholipase A2 blocks malaria parasite development in the mosquito midgut by inhibiting ookinete association with the midgut surface
1 Medical Entomology Section,
2 Malaria Vaccines Section and
3 Biophysical Parasitology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425, USA
Correspondence *Present address: Chromatin Inc., 2201 West Campbell Park Drive, Chicago, IL 60612, USA (e-mail: zieler{at}chromatininc.com)
Accepted September 20, 2001
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Summary |
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Key words: malaria, ookinete, transmission blocking, phospholipase, p-bromophenacyl bromide, eastern diamondback rattlesnake, Crotalus adamanteus, Plasmodium gallinaceum, Plasmodium falciparum, Aedes aegypti.
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Introduction |
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The malaria parasite begins its development shortly after the mosquito ingests an infected blood meal. Male and female gametes emerge from infected red blood cells and mate to form a zygote. Over the next 1624 h, the zygote transforms into a motile ookinete, which then leaves the blood meal, penetrates through the chitinous peritrophic membrane, makes contact with the midgut epithelium and invades a midgut cell. It crosses the epithelium until it reaches the basement membrane on the other side of the midgut wall and rounds up against this structure to form an oocyst. The oocyst enlarges and produces numerous sporozoites. These are released and migrate to, and invade, the salivary glands, from where they are injected into a new vertebrate during blood-feeding on the host.
Little is known about how the parasite recognizes and invades the mosquito midgut epithelium. Studies using light and electron microscopy have shed some light on the events occurring between ookinete invasion of the midgut epithelium and oocyst formation (Mehlhorn et al., 1980; Meis and Ponnudurai, 1987
; Meis et al., 1989
; Sieber et al., 1991
; Syafruddin et al., 1991
; Torii et al., 1992
). Recently, the establishment of an in vitro system has allowed more detailed studies of the interactions between Plasmodium gallinaceum ookinetes and the midgut of Aedes aegypti (Shahabuddin et al., 1997
; Shahabuddin and Pimenta, 1998
; Zieler et al., 1998
, 1999
). In vitro, the ookinetes bind to the microvillated midgut surface and invade midgut cells, reflecting their normal behavior in the mosquito host (Shahabuddin et al., 1997
; Shahabuddin and Pimenta, 1998
). Using these novel methods, we and others have been able to observe specific adhesion of ookinetes to the mosquito midgut (Shahabuddin et al., 1997
; Zieler et al., 1999
), invasion of the epithelium (Zieler and Dvorak, 2000
), changes in the morphology of the invaded cells (Shahabuddin and Pimenta, 1998
) and the resulting death of the invaded cells (Zieler and Dvorak, 2000
; Han et al., 2000
).
During the course of our attempts to understand the binding of ookinetes to the midgut surface, we discovered that a phospholipase A2 (PLA2) from the eastern diamondback rattlesnake (Crotalus adamanteus) inhibits ookinete adhesion and oocyst formation. The C. adamateus PLA2 belongs to a large and well-studied class of highly conserved secreted PLA2s that are commonly found in snake and insect venoms and in vertebrate pancreatic excretions (for reviews, see Slotboom et al., 1982; Waite, 1987
). These enzymes are small (approximately 15 kDa) and extremely stable because of the presence of seven disulfide bridges in the globular structures. PLA2s hydrolyze stereospecifically the acyl ester in position 2 of 3-sn-phosphoglycerides to form lysophospholipids and fatty acids, causing the degradation of phospholipids in membrane bilayers.
Although they are highly water-soluble, PLA2s are much more active towards aggregated phospholipid present in micelles, monolayers or natural or artificial bilayers than towards dispersed monomeric substrates (Dennis, 1973, 1983
; Verger et al., 1973
; Hønger et al., 1997
). The reason for this preference is the ability of PLA2s to interact efficiently with membranes and insert into the lipid bilayer to bind to their substrates (interfacial binding). Several amino acid residues found on the enzyme surface participate in interfacial binding in a manner independent of hydrolytic activity (Keith et al., 1981
; Brunie et al., 1985
; White et al., 1990
; Jain et al., 1995
; Gelb et al., 1999
). Because of their preference for the interfacial membrane environment, PLA2s are also very sensitive to the physical state of membranes, and their binding is strongly influenced by lateral lipid pressure, lipid composition, surface charge, membrane curvature, temperature and the presence of gel or fluid lipid phases in the membrane (op den Kamp et al., 1974
; Kensil and Dennis, 1979
; Goormaghtigh et al., 1981
; Apiz-Castro et al., 1982
; Dawson et al., 1984
; Thuren et al., 1987
; Burack et al., 1993
; Lehtonen and Kinunnen, 1995
; Hønger et al., 1997
). Most PLA2s prefer to bind to membranes perturbed by a variety of factors that cause lipid packing defects. Furthermore, the PLA2s are very diverse in terms of their ability to penetrate lipid bilayers, which has made them into useful tools for studying lateral lipid pressures in biological membranes (Demel et al., 1975
; Waite, 1987
).
Because of their interfacial binding characteristics, PLA2s bind efficiently to aggregated lipids even in the absence of enzymatic activity (Tinker et al., 1980; Condrea et al., 1981
; Kini and Evans, 1989
). Studies of this property have benefited from the use of p-bromophenacyl bromide (pBPB), which reacts irreversibly with an essential histidine residue in the active site of the enzyme (Volwerk et al., 1974
). It has been found that pBPB-inactivated PLA2s display the same binding characteristics as the catalytically active enzyme. As a result, many of the venom PLA2s retain some or all of their toxic effects in animals after inactivation with pBPB (for reviews, see Rosenberg, 1986
; Kini and Evans, 1989
).
Because of their binding properties, small size, stability and heterogeneity, the PLA2s represent a useful and convenient tool for probing interactions between cell surfaces. We report here that a snake venom PLA2 inhibits oocyst formation of malaria parasites by blocking the association between ookinetes and the midgut surface. We propose that the gene coding for this enzyme may be useful as a genetic tool for interfering with malaria parasite development in mosquitoes.
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Materials and methods |
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Chickens, mosquitoes and parasites
The 8A strain of Plasmodium gallinaceum was used throughout this study. Parasites were maintained in white Leghorn chickens by serial blood passage. Mosquitoes were raised and fed using standard techniques (Gerberg et al., 1994; Higgs and Beaty, 1996
). Zygotes were purified from the blood of infected chickens as described in detail elsewhere (Zieler et al., 1999
). After purification of zygotes, the parasites were resuspended in M199 medium supplemented with 2 mmol l1 L-glutamine, 100 units ml1 penicillin and 100 µg ml1 streptomycin (ookinete medium) at a density of 5x106 zygotes ml1 and incubated for 1624 h at 25°C to allow development of ookinetes. Oocyst formation assays and transmission-blocking assays (Sieber et al., 1991
; Higgs and Beaty, 1996
) were performed as described previously (Zieler et al., 1999
). Oocyst numbers in infected mosquitoes were compared amongst samples using MannWhitney rank sum analysis.
Purification to homogeneity of commercially available Crotalus adamanteus PLA2
The C. adamanteus PLA2 used in all procedures was bought from Sigma-Aldrich and further purified to homogeneity by a single step of reversed-phase high-performance liquid chromatography (HPLC). The enzyme (12 mg, approximately 80 % pure) was dissolved in 20 % acetonitrile, 0.1 % trifluoroacetic acid (TFA) and injected into an Alltech Macrosphere C18 column (30 nm, 250 mmx4.6 mm) equilibrated with 20 % acetonitrile, 0.1 % TFA, run at a flow rate of 0.5 ml min1 with a 90 min gradient from 20 % to 60 % acetonitrile, 0.1 % TFA, with collection of 0.5 ml fractions. The PLA2 was the major peak, eluting at approximately 6065 min. The fractions containing PLA2 activity were pooled and dried under vacuum in a SpeedVac. The protein was redissolved in 100 mmol l1 Hepes, pH 7.5, and dialyzed extensively against 1 mmol l1 Hepes, pH 7.5, in a Slide-a-Lyzer dialysis cassette (3.5 kDa molecular mass cut-off).
Labeling of Crotalus adamateus PLA2 with Alexa 488
Since PLA2s are generally sensitive to reaction with amine-reactive compounds (Wells, 1973; Hazlett and Dennis, 1985
), the PLA2 was fluorescently labeled using a technique that modifies carboxylate groups in the protein (Staros et al., 1986
; Grabarek and Gergely, 1990
). These carboxylate groups are not thought to be involved in interfacial binding of the enzyme (Fleer et al., 1981
; Waite, 1987
; Gelb et al., 1999
). The active-site aspartate was protected from modification by addition of calcium to the reaction (Fleer et al., 1981
). Coupling of Alexa 488 (Molecular Probes, Eugene, OR, USA) to PLA2 was performed at room temperature (approximately 23°C) in 0.1 mol l1 MES, 0.5 mol l1 NaCl, 1 mol l1 CaCl2, pH 6.0. An enzyme solution of 1 mg ml1 was prepared in this buffer; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Sigma) was added to a final concentration of 2 mmol l1 and N-hydroxysulfosuccinimide (sulfo-NHS; Pierce) was added to a final concentration of 5 mmol l1. After 15 min at 23°C, the reaction was stopped by the addition of 2-mercaptoethanol to a final concentration of 20 mmol l1. Alexa 488 hydrazide (Molecular Probes) was then added to a final concentration of 2 mmol l1, the pH of the mixture was raised to 7.0, and the reaction was allowed to proceed for 2 h at room temperature (Staros et al., 1986
; Grabarek and Gergely, 1990
). Finally, hydroxylamine (Sigma) was added to a final concentration of 50 mmol l1 and was allowed to react at room temperature for 3 h.
The modified enzyme was desalted by dialysis in a Slide-A-Lyzer dialysis cassette (3.5 kDa molecular mass cut-off; Pierce), followed by purification over a SepPak Plus C18 cartridge (Waters Corporation, Milford, MA, USA) and re-dialysis. Spectophotometric and fluorescence determinations showed an approximately 2:1 ratio of enzyme molecule to dye molecule (one dye molecule per enzyme dimer). This was determined by measuring the approximate enzyme concentration by absorbance at 280 nm, establishing a standard fluorescence curve of serial dilutions of a known concentration of the Alexa 488 dye and correlating the observed fluorescence of the labeled enzyme sample with the standard curve.
Staining of midguts and ookinetes with fluorescent PLA2
Midgut epithelia or ookinetes were suspended in ookinete medium +10 % heat-inactivated chicken serum (adhesion medium). Fluorescently labeled PLA2 was added to a final concentration of 2.9 µmol l1, and the samples were incubated for 10 min at room temperature. The samples were then washed twice with 1 ml of adhesion medium, transferred to a glass slide, spread out and fixed with 4 % paraformaldehyde, 2.5 % glutaraldehyde in phosphate-buffered saline (PBS). The fixed cells or midguts were transferred to a fresh glass slide, covered with a coverslip and examined by widefield fluorescence microscopy. Alternatively, midguts or ookinetes were examined unfixed by fluorescence microscopy, with similar results being obtained in both cases. The fluorescence of stained, unfixed midguts was also measured in a Fluorolite 1000 fluorimeter (Dynatech Laboratories, Chantilly, VA, USA), which confirmed the observation of PLA2 binding to midguts.
Preparation of isolated midgut epithelia, staining of ookinetes with PKH26 and ookinete/midgut adhesion assay
These procedures were performed as described in previous publications (Shahabuddin et al., 1997; Zieler et al., 1999
). Briefly, midguts were removed from female mosquitoes 24 h after a blood meal, cut in half with a scalpel blade, and the two half-midgut epithelia (midgut sheets) were separated from the blood meal. Each pair of midgut sheets was transferred into a separate Eppendorf tube containing adhesion medium. For pre-treatment with PLA2, the midguts were mixed with 20 µl of adhesion medium containing a known quantity of PLA2 and incubated at room temperature for 10 min, while control midguts were incubated with adhesion medium only. After the incubation, 80 µl of adhesion medium was added to each tube to dilute the PLA2, followed by 50 µl of ookinetes stained with PKH26 and suspended in adhesion medium (roughly 3x105 to 106 ookinetes added to each pair of midgut sheets). Staining of ookinetes with PKH26 (Sigma) was performed by diluting the PKH26 solution supplied by Sigma 1:40 in 5.4 % glucose, using this solution to resuspend a pellet of ookinetes, incubating the parasites for 30 s and then washing them three times with adhesion medium by successive centrifugation and resuspension to remove excess PKH26. Adhesion of ookinetes to the midguts was achieved by centrifuging the tubes containing midgut sheets and parasites three times at 300 g in a microfuge and resuspending the midgut sheets and parasites in each tube after each centrifugation by flicking the tube. The midgut sheets were then washed twice with 1 ml of adhesion medium, spread out flat on glass slides with the luminal side facing up, fixed by the addition of 2.5 % glutaraldehyde, 4 % paraformaldehyde in PBS, and examined by fluorescence microscopy to count the bound ookinetes. All adhesion experiments were performed with quadruplicate samples. Statistical analyses of differences between sample groups were performed using the t-test.
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Results |
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When fed to Aedes aegypti mosquitoes as part of an infected blood meal, the PLA2 strongly inhibits oocyst formation (Table 1). This effect does not require the hydrolytic activity of the enzyme; when PLA2 was preincubated with the irreversible inhibitor p-bromophenacyl bromide (pBPB), oocyst formation was still strongly reduced. The inhibitor alone had some effect on oocyst numbers (Table 1). However, the enzyme in the presence of the inhibitor was completely inactive in our assays. The effect of the PLA2 is concentration-dependent (Fig. 1) both in the presence and in the absence of pBPB. We determined that the lowest concentration of phospholipase that resulted in a statistically significant reduction in oocyst frequency when feeding infected blood is 21 nmol l1 (Fig. 1). The PLA2 was also fed to Anopheles gambiae and Anopheles stephensi together with cultured gametocytes of the human malaria parasite Plasmodium falciparum. Similar levels of inhibition were seen in both anopheline species (Table 1), and the concentration-dependence of PLA2 inhibition was similar to that observed in P. gallinaceum.
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PLA2 has no effect on ookinete development or viability
To determine whether any pre-ookinete developmental stage is sensitive to the PLA2, we added the enzyme during the course of exflagellation and purification of macrogametes and zygotes during routine preparation of parasites from infected chicken blood. There was no difference in the number of exflagellation events in the presence or absence of PLA2, nor did the PLA2 have a significant effect on the total yield of macrogametes/zygotes obtained at the end of the purification (Table 2). The ookinete yields from the treated parasites were also unaffected. When the PLA2 was added to purified zygotes, which were then incubated in ookinete medium (for 24 h) to allow transformation into ookinetes, there was no effect on ookinete development in terms of either frequency (Table 3) or timing (data not shown). Some of the samples listed in Table 3 had PLA2 present continuously from exflagellation through ookinete formation and showed no significant differences in ookinete numbers compared with the controls. Furthermore, the ookinetes in all the samples displayed normal motility on glass slides. We conclude from these data that the PLA2 does not affect parasite development in the blood meal.
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The adhesion assays performed in the presence and absence of PLA2 allowed us to observe shedding of lipids from the posterior ends of ookinetes, which can be detected as the capping of fluorescent stain on the posterior end of the parasite, and which correlates with ookinete motility. In certain ookinete preparations, all parasites bound to the midgut surface show capping after the adhesion is complete and the fixed midguts are being examined for the number of bound ookinetes. In 10 pairs of midgut sheets where capping was observed, there were never any visible differences in the posterior capping of fluorescent stains in the presence or absence of PLA2. It is likely, therefore, that the PLA2 does not interfere with ookinete motility.
Adhesion assays using PLA2s from other organisms were used to determine whether the ability of the C. adamanteus enzyme to inhibit ookinete/midgut binding is a general feature of PLA2s. As shown in Fig. 3, PLA2s from the venoms of various snakes, as well as honeybee venom PLA2 and mammalian pancreatic PLA2s, were used to pretreat midguts in adhesion assays. The results (Fig. 3) indicate that the PLA2s derived from snake and insect venoms strongly inhibit ookinete binding, while the mammalian pancreatic enzymes do not. The extent of inhibition of oocyst formation by the different PLA2s in oocyst formation assays mirrors their inhibition of in vitro adhesion (data not shown).
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The labeled PLA2 was used to stain isolated midguts and ookinetes. Staining was performed in the presence of 10 % chicken serum, as a non-specific competitor, and the stained tissues or cells were washed extensively after staining to remove non-specifically bound PLA2. The protein readily bound to midgut epithelia and showed a diffuse staining on both the luminal surface and the basement membrane on the other side of the epithelium (Fig. 4). The staining of the luminal surface was often irregular, with certain areas or individual cells staining more brightly for unknown reasons. Higher magnifications clearly showed surface staining of the midgut cells (Fig. 4D), although the lack of resolution made it difficult to determine whether the PLA2 was associating with the microvilli-associated network observed on the midgut surface in earlier studies (Zieler et al., 1998, 2000
). Although the unstained midguts are not shown for comparison, the degree of autofluorescence was low compared with the stained midgut and resulted in a very dim image at the lamp intensity and gain settings used for the digital camera. We also demonstrated that the binding of labeled PLA2 to the midguts could be inhibited by an excess of unlabeled enzyme. In contrast to the midgut staining by the labeled PLA2, however, labeled PLA2 completely failed to bind to ookinetes. In addition, the PLA2 failed to label isolated peritrophic membranes, suggesting that its ligand is found only on the midgut surface (data not shown).
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Discussion |
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PLA2s from the venoms of other snakes and from honeybees appear to have the same property as the PLA2 from Crotalus adamanteus (see Fig. 3). In contrast, the mammalian pancreatic PLA2s do not efficiently block adhesion. The data in Fig. 3 suggest a correlation between the membrane-inserting abilities of a PLA2 and its ability to block ookinete adhesion and oocyst formation. The mammalian pancreatic PLA2s tend to have a very low membrane-inserting ability, while the honeybee venom PLA2 has the highest inserting ability measured in any phospholipase (Demel et al., 1975; Waite, 1987
). This result is consistent with our finding that the hydrolytic activity of the PLA2 is not an essential part of its antiparasitic activity and suggests that the inhibition of ookinete adhesion by the PLA2 is primarily dependent on the enzymes property of binding to exposed membrane lipids.
We have previously reported a novel membranous structure on the midgut surface that we have termed the microvilli-associated network (MN) and which seems to be the primary structure with which ookinetes associate on the midgut surface (Zieler et al., 1998, 2000
). It is likely that the PLA2 binds to the MN. Unfortunately, this structure is difficult to see using light microscopy, and in our fluorescence labeling experiments we were unable to determine whether the labeled PLA2 does indeed bind to MN strands.
The inhibition by the PLA2 of ookinete association with the midgut surface could be a result of its direct binding to the same periodate-sensitive carbohydrate ligand (Zieler et al., 1999) recognized by the ookinete. In this case, the ookinete ligand would probably be a glycolipid or a highly periodate-sensitive phospholipid such as phosphatidylglycerol. We were unable to test whether periodate treatment of midguts affects PLA2 binding, since extensive cell damage occurs during periodate treatment and the PLA2 binds more efficiently to the perturbed membranes in damaged cells (Dawson et al., 1984
; Thuren et al., 1987
).
In contrast, it is unlikely that inhibition of ookinete adhesion by PLA2s is related to their ability to bind to heparin and other anionic glycosaminoglycans. First, this property of PLA2s is best-documented in the pancreatic enzymes (Diccianni et al., 1990, 1991
), which are not very active in our assays. Second, we have tried heparin, as well as a variety of other anionic glycosaminoglycans, as competitors in adhesion assays and have been unable to detect any inhibition at concentrations as high as 500 µg ml1. Third, the Crotalus adamanteus PLA2 inhibits ookinete adhesion even in the presence of heparin (data not shown).
In the adhesion assay, we also tested human annexin V, a protein known to bind to acidic phospholipids (such as phosphatidylserine) normally present in the inner leaflet of the plasma membrane bilayer (Swairjo et al., 1995; Reutlingsperger and van Heerde, 1996
), and found no inhibition of adhesion. Annexins are Ca2+-dependent phospholipid-binding proteins that compete with PLA2s for binding to membranes (Davidson et al., 1987
; Buckland and Wilton, 1998a
,b
). The phospholipid preference of PLA2s, or the manner in which they disrupt lipid bilayers during binding, is probably an important characteristic that enables these enzymes specifically to disrupt ookinete/midgut interactions.
The PLA2s have a number of unique properties that make them especially suitable for expression in mosquito midguts as refractory genes. First, they are exceptionally stable and resistant to protease cleavage, an important property in the degradative environment of the midgut lumen. Second, they are generally secreted as proenzymes, which are activated by trypsin cleavage (Pieterson et al., 1974; Waite, 1987
). Proenzyme activation will occur after secretion into the midgut lumen, as happens with midgut chitinases (Shen and Jacobs-Lorena, 1997
). The feasibility of expressing PLA2 enzymes as proenzymes in a variety of heterologous expression systems has already been demonstrated (Verheij and de Haas, 1991
). Third, the hydrolytic activity of PLA2s is not required for inhibition of parasite development, implying that the midgut-secreted enzyme can be catalytically inactivated by mutation if necessary. We have shown with membrane feeds that ingestion of the active PLA2 has no measurable effect on the female mosquitoes (Table 6), arguing that a midgut-expressed PLA2 would be well-tolerated by transgenic mosquitoes. PLA2-expressing transgenic Aedes aegypti are currently being generated using the recently developed transformation methods for this organism (Coates et al., 1998
; Jasinskiene et al., 1998
). The PLA2 gene will thus be one of the first genes to be tested in transgenic mosquitoes for its ability to confer refractoriness to malaria parasites.
Our long-term studies are aimed at a complete characterization of the interaction between malaria parasites and mosquito tissues. In the process of this work, we will further our understanding of the biology of this parasite, and we hope that this knowledge will be a starting point for developing new methods of malaria control.
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Acknowledgments |
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References |
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---|
Apiz-Castro, R., Jain, M. K. and de Haas, G. H. (1982). Origin of the latency phase during the action of phospholipase A2 on unmodified phosphatidylcholine vesicles. Biochim. Biophys. Acta 688, 349356.[Medline]
Brunie, S., Bolin, J., Gewirth, D. and Sigler, P. B. (1985). The refined crystal structure of dimeric phospholipase A2 at 2 Å. J. Biol. Chem. 260, 97429749.
Buckland, A. G. and Wilton, D. C. (1998a). Inhibition of human cytosolic phospholipase A2 by human annexin V. Biochem. J. 329, 369372.[Medline]
Buckland, A. G. and Wilton, D. C. (1998b). Inhibition of phospholipase A2 by annexin. V. Competition for anionic phospholipid interfaces allows an assessment of the relative interfacial affinities of secreted phospholipases A2. Biochim. Biophys. Acta 1391, 367376.[Medline]
Burack, W. R., Yuan, Q. and Biltonen, R. L. (1993). Role of lateral phase separation in the modulation of phospholipase A2 activity. Biochemistry 32, 583589.[Medline]
Coates, C., Jasinskiene, N., Miyashiro, L. and James, A. A. (1998). Mariner transposition and transformation of the yellow fever mosquito, Aedes aegypti. Proc. Natl. Acad. Sci. USA 95, 37483751.
Condrea, E., Yang, C. C. and Rosenberg, P. (1981). Lack of correlation between anticoagulant activity and phospholipid hydrolysis by snake venom phospholipases A2. Thromb. Haemost. 45, 8285.[Medline]
Crampton, J. M., Warren, A., Lycett, G. J., Hughes, M. A., Comley, I. P. and Eggleston, P. (1994). Genetic manipulation of insect vectors as a strategy for the control of vector-borne disease. Ann. Trop. Med. Parasitol. 88, 312.[Medline]
Davidson, F. F., Dennis, E. A., Powell, M. and Glenney, J. R. J. (1987). Inhibition of phospholipase A2 by lipocortins and calpactins. An effect of binding to substrate phospholipids. J. Biol. Chem. 262, 16981705.
Dawson, R. M. C., Irvine, R. F., Bray, J. and Quinn, P. J. (1984). Long-chain unsaturated diacylglycerols cause a perturbation in the structure of phospholipid bilayers rendering them susceptible to phospholipase attack. Biochem. Biophys. Res. Commun. 125, 836842.[Medline]
Demel, R. A., Geurts van Kessel, W. S. M., Zwaal, R. F. A., Roelofsen, B. and van Deenen, L. M. (1975). Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers. Biochim. Biophys. Acta 406, 97107.[Medline]
Dennis, E. A. (1973). Phospholipase A2 activity towards phosphatidylcholine in mixed micelles: surface dilution kinetics and the effect of thermotropic phase transitions. Arch. Biochem. Biophys. 158, 485493.[Medline]
Dennis, E. A. (1983). Phospholipases. In The Enzymes, 3rd edn, vol. 16 (ed. P. D. Boyer), pp. 307353. New York: Academic Press.
Diccianni, M. B., Lilly-Staudermann, M., McLean, L. R., Balasubramaniam, A. and Harmony, J. A. K. (1991). Heparin prevents the binding of phospholipase A2 to phospholipid micelles: importance of the amino-terminus. Biochemistry 30, 90909097.[Medline]
Diccianni, M. B., Mistry, M. J., Hug, K. and Harmony, J. A. K. (1990). Inhibition of phospholipase A2 by heparin. Biochim. Biophys. Acta 1046, 242248.[Medline]
Fleer, E. A., Verheij, H. M. and de Haas, G. H. (1981). Modification of carboxylate groups in bovine pancreatic phospholipase A2. Identification of aspartate-49 as Ca2+-binding ligand. Eur. J. Biochem. 113, 283288.[Abstract]
Gelb, M. H., Cho, W. and Wilton, D. C. (1999). Interfacial binding of secreted phospholipase A2: more than electrostatics and a major role for tryptophan. Curr. Opin. Struct. Biol. 9, 428432.[Medline]
Gerberg, E. J., Barnard, D. R. and Ward, R. A. (1994). Manual for Mosquito Rearing and Experimental Techniques. Lake Charles, LA: American Mosquito Control Association.
Goormaghtigh, E., van Campenhoud, M. and Ruysschaert, J.-M. (1981). Lipid phase separation mediates binding of porcine pancreatic phospholipase A2 to its substrate. Biochem. Biophys. Res. Commun. 101, 14101418.[Medline]
Grabarek, Z. and Gergely, J. (1990). Zero-length cross-linking procedure with the use of active esters. Anal. Biochem. 185, 131135.[Medline]
Gwadz, R. W. (1994). Genetic approaches to malaria control: how long the road? Am. J. Trop. Med. Hyg. 50, 116125.[Medline]
Han, Y. S., Thompson, J., Kafatos, F. C. and Barillas-Mury, C. (2000). Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J. 19, 60306040.
Hazlett, T. L. and Dennis, E. A. (1985). Aggregation studies on fluorescein-coupled cobra venom phospholipase A2. Biochemistry 24, 61526158.[Medline]
Higgs, S. and Beaty, B. J. (1996). Rearing and containment of mosquito vectors. In The Biology of Disease Vectors (ed. B. J. Beaty and W. C. Marquardt), pp. 595605. Niwot, CO: University Press of Colorado.
Hønger, T., Jørgensen, K., Stokes, D., Biltonen, R. L. and Mouritsen, O. G. (1997). Phospholipase A2 activity and physical properties of lipid-bilayer substrates. Meth. Enzymol. 286, 168190.[Medline]
Jain, M. K., Gelb, M. H., Rogers, J. and Berg, O. G. (1995). Kinetic basis for interfacial catalysis by phospholipase A2. Meth. Enzymol. 249, 567614.[Medline]
Jasinskiene, N., Coates, C. J., Benedict, M. Q., Cornel, A. J., Rafferty, C. S., James, A. A. and Collins, F. H. (1998). Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly. Proc. Natl. Acad. Sci. USA 95, 37433747.
Kaneshiro, E. S., Wyder, M. A., Wu, Y.-P. and Cushion, M. T. (1993). Reliability of calcein acetoxy methyl ester and ethidium homodimer or propidium iodide for viability assessment of microbes. J. Microbiol. Meth. 17, 116.
Keith, C., Feldman, D. S., Deganello, S., Glick, J., Ward, K. B., Jones, E. O. and Sigler, P. B. (1981). The 2.5 Å crystal structure of a dimeric phospholipase A2 from the venom of Crotalus atrox. J. Biol. Chem. 256, 86028607.
Kensil, C. R. and Dennis, E. A. (1979). Action of cobra venom phospholipase A2 on the gel and liquid crystalline states of dimyristoyl phosphatidylcholine vesicles. J. Biol. Chem. 254, 58435848.[Abstract]
Kini, R. M. and Evans, H. J. (1989). A model to explain the pharmacological effects of snake venom phopholipase A2. Toxicon 27, 613635.[Medline]
Lehtonen, J. Y. A. and Kinunnen, P. K. J. (1995). Phospholipase A2 as a mechanosensor. Biophys. J. 68, 18881894.[Abstract]
Mehlhorn, H., Peters, W. and Haberkorn, A. (1980). The formation of kinetes and oocyst in Plasmodium gallinaceum (Haemosporidia) and considerations on phylogenetic relationships between Haemosporidia, Piroplasmida and other coccidia. Protistologica 16, 135154.
Meis, J. F. and Ponnudurai, T. (1987). Ultrastructural studies on the interaction of Plasmodium falciparum ookinetes with the midgut epithelium of Anopheles stephensi mosquitoes. Parasitol. Res. 73, 500506.[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, 1319.[Medline]
op den Kamp, J. A. F., de Gier, J. and van Deenen, L. L. M. (1974). Hydrolysis of phosphatidylcholine liposomes by pancreatic phospholipase A2 at the transition temperature. Biochim. Biophys. Acta 345, 253256.[Medline]
Papadopoulos, N. G., Dedoussis, G. V. Z., Spanakos, G., Gritzapis, A. D., Baxevanis, C. N. and Papamichail, M. (1994). An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry. J. Immunol. Meth. 177, 101111.[Medline]
Pieterson, W. A., Vidal, J. C., Volwerk, J. J. and de Haas, G. H. (1974). Zymogen-catalyzed hydrolysis of monomeric substrates and the presence of a recognition site for lipidwater interfaces in phospholipase A2. Biochemistry 13, 14551460.[Medline]
Reutlingsperger, C. P. M. and van Heerde, W. L. (1996). Annexin V and cell surface-expressed phosphatidylserine: a revealing pas de deux. In Annexins: Molecular Structure to Cellular Function (ed. B. A. Seaton), pp. 201211. Austin, TX: Chapman & Hall.
Rosenberg, P. (1986). The relationship between enzymatic activities and pharmacological properties of phospholipases in natural poisons. In Natural Toxins: Animal, Plant and Microbial (ed. J. B. Harris), pp. 129174. Oxford: Clarendon Press.
Shahabuddin, M., Fields, I., Bulet, P., Hoffmann, J. A. and Miller, L. H. (1998). Plasmodium gallinaceum: differential killing of some mosquito stages of the parasite by insect defensin. Exp. Parasitol. 89, 103112.[Medline]
Shahabuddin, M. and Pimenta, P. F. (1998). Plasmodium gallinaceum preferentially invades vesicular ATPase-expressing cells in Aedes aegypti midgut. Proc. Natl. Acad. Sci. USA 95, 33853389.
Shahabuddin, S., Gayle, M., Zieler, H. and Laughinghouse, A. (1997). Plasmodium gallinaceum: fluorescent staining of zygotes and ookinetes to study malaria parasites in the mosquito. Exp. Parasitol. 88, 7984.
Shen, Z. and Jacobs-Lorena, M. (1997). Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae. J. Biol. Chem. 272, 2889528900.
Sieber, K. P., Huber, M., Kaslow, D., Banks, S. M., Torii, M., Aikawa, M. and Miller, L. H. (1991). The peritrophic membrane as a barrier: its penetration by Plasmodium gallinaceum and the effect of a monoclonal antibody to ookinetes. Exp. Parasitol. 72, 145156.[Medline]
Slotboom, A. J., Verheij, H. M. and de Haas, G. H. (1982). On the mechanism of phospholipase A2. In Phospholipids (ed. J. N. Hawthorne and G. B. Ansell), pp. 359434. Amsterdam: Elsevier Biomedical Press.
Staros, J. V., Wright, R. W. and Swingle, D. M. (1986). Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem. 156, 220222.[Medline]
Swairjo, M. A., Concha, N. O., Kaetzel, M. A., Dedman, J. R. and Seaton, B. A. (1995). Ca2+-bridging mechanism and phospholipid head group recognition in the membrane-binding protein annexin V. Nature Struct. Biol. 2, 968974.[Medline]
Syafruddin, Arakawa, R., Kamimura, K. and Kawamoto, F. (1991). Penetration of the mosquito midgut wall by the ookinetes of Plasmodium yoelii nigeriensis. Parasitol. Res. 77, 230236.[Medline]
Thuren, T., Tulkki, J. A., Virtanen, J. A. and Kinnunen, P. K. J. (1987). Triggering of the activity of phospholipase A2 by an electric field. Biochemistry 26, 49074910.[Medline]
Tinker, D. O., Low, R. and Lucassen, M. (1980). Heterogeneous catalysis by phospholipase A2: mechanism of hydrolysis of gel phase phosphatidylcholine. Can. J. Biochem. 58, 898912.[Medline]
Torii, M., Nakamura, K., Sieber, K. P., Miller, L. H. and Aikawa, M. (1992). Penetration of the mosquito (Aedes aegypti) midgut wall by the ookinetes of Plasmodium gallinaceum. J. Protozool. 39, 449454.[Medline]
Verger, R., Mieras, M. C. E. and de Haas, G. H. (1973). Action of phospholipase A at interfaces. J. Biol. Chem. 248, 40234034.
Verheij, H. M. and de Haas, G. H. (1991). Cloning, expression and purification of porcine pancreatic phospholipase A2 and mutants. Meth. Enzymol. 197, 214223.[Medline]
Volwerk, J. J., Pieterson, W. A. and de Haas, G. H. (1974). Histidine at the active site of phospholipase A2. Biochemistry 13, 14461454.[Medline]
Waite, M. (1987). The Phospholipases. New York: Plenum Press.
Wells, M. A. (1973). Effects of chemical modification on the activity of Crotalus adamanteus phospholipase A2. Evidence for an essential amino group. Biochemistry 12, 10861093.[Medline]
White, S. P., Scott, D. L., Otwinowski, Z., Gelb, M. H. and Sigler, P. B. (1990). Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue. Science 250, 15601563.[Medline]
Zieler, H. and Dvorak, J. A. (2000). Invasion in vitro of mosquito midgut cells by the malaria parasite proceeds by a conserved mechanism and results in death of the invaded midgut cells. Proc. Natl. Acad. Sci. USA 97, 1151611521.
Zieler, H., Garon, C. F., Fischer, E. R. and Shahabuddin, M. (1998). Adhesion of Plasmodium gallinaceum ookinetes to the Aedes aegypti midgut: sites of parasite attachment and morphological changes in the ookinete. J. Euk. Microbiol. 45, 512520.[Medline]
Zieler, H., Garon, C. F., Fischer, E. R. and Shahabuddin, M. (2000). A tubular network associated with the brush-border surface of the Aedes aegypti midgut: implications for pathogen transmission by mosquitoes. J. Exp. Biol. 203, 15991611.
Zieler, H., Nawrocki, J. P. and Shahabuddin, M. (1999). Plasmodium gallinaceum ookinetes adhere specifically to the midgut epithelium of Aedes aegypti by interaction with a carbohydrate ligand. J. Exp. Biol. 202, 485495.