QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zieler, H. Articles by Ribeiro, J. M. C. Search for Related Content PubMed PubMed Citation Articles by Zieler, H. Articles by Ribeiro, J. M. C.

A snake venom phospholipase A₂ blocks malaria parasite development in the mosquito midgut by inhibiting ookinete association with the midgut surface

Helge Zieler^1,*, David B. Keister², James A. Dvorak³ and José M. C. Ribeiro¹

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

View larger version (16K): [in a new window]   Fig. 1. Oocyst numbers from membrane feeds of purified Crotalus adamanteus PLA2. Open circles, the PLA2 was mixed with washed, infected chicken blood (16 % parasitemia) and fed to mosquitoes. Open triangles, the PLA2 was pretreated with p-bromophenacyl bromide (pBPB) at a final concentration of 100 µmol l–1 and then mixed with washed, infected chicken blood (16 % parasitemia) and fed to mosquitoes. Filled circles, the PLA2 was added to an ookinete suspension in naive chicken blood and fed to mosquitoes. Filled triangles, the PLA2 was pretreated with pBPB, then added to an ookinete suspension in naive chicken blood and fed to mosquitoes. Oocyst numbers were scored 8 days after the membrane feed. Each data point represents the geometric mean of oocyst numbers from at least 20 mosquitoes expressed as percentage inhibition relative to control mosquitoes fed either with infected blood plus buffer or with infected blood plus inhibitor alone.

View larger version (17K): [in a new window]   Fig. 2. Midguts were pretreated with 3.2 µmol l–1 PLA2 in M199 medium for 10 min, and the treated guts were then used in adhesion assays. Control, midguts treated with buffer only; PLA2 alone, midguts treated with active PLA2; PLA2+pBPB, midguts treated with PLA2 in the presence of 100 µmol l–1 p-bromophenacyl bromide (pBPB), an inhibitor of the enzyme; pBPB alone, midguts pretreated with the inhibitor pBPB only; PLA2+ZnCl2, midguts pretreated with PLA2 in the presence of 100 µmol l–1 ZnCl2, an inhibitor of the enzyme; ZnCl2 alone, midguts treated with 100 µmol l–1 ZnCl2. Each column is the mean of 1–2 experiments, each performed in quadruplicate. The error bars represent ± S.E.M. (N=4–8). P values for each column are as follows: PLA2 alone, P=0.0002; PLA2+pBPB, P<0.0001; pBPB alone, P=0.98; PLA2+ZnCl2, P=0.004; ZnCl2 alone, P=0.91.

View larger version (24K): [in a new window]   Fig. 3. Midguts were pretreated with different PLA2s (final concentration 10 µmol l–1) in M199 medium for 10 min and then used in adhesion assays. Control, midguts treated with buffer only. The PLA2s used in treatments were as follows: Bovine pancr., bovine pancreatic PLA2; Porcine pancr., porcine pancreatic PLA2; Bee venom, honeybee venom PLA2; Crotalus d. t., Crotalus durissus terrificus venom PLA2 (crotoxin); Crotalus ad., Crotalus adamanteus venom PLA2 (7 µmol l–1); Naja moss. I, Naja mossambica mossambica venom PLA2 isozyme I (pI=6.5); Naja moss. II, Naja mossambica mossambica venom PLA2 isozyme II (pI=8.8); Naja moss. III, Naja mossambica mossambica venom PLA2 isozyme III (pI=9.6); Naja naja, Naja naja venom PLA2. The PLA2s were dialyzed extensively before use in the assays. Each column represents the results of a single experiment, performed in quadruplicate. Error bars represent ± S.E.M. (N=4). P values for each column are as follows: Bovine pancr., P=0.23; Porcine pancr., P=0.05; Bee venom, P=0.001; Crotalus d. t., P=0.001; Crotalus ad., P=0.002; Naja moss. I, P=0.003; Naja moss. II, P=0.004; Naja moss. III, P=0.02; Naja naja, P=0.003.

View larger version (113K): [in a new window]   Fig. 4. Bright-field (A,C) and fluorescence (B,D) images of an Aedes aegypti blood-fed midgut sheet stained with fluorescently labeled Crotalus adamanteus PLA2 and fixed. The images in C and D are a higher magnification of the upper portion of the midgut shown in A and B. Scale bars: A, 500 µm; C, 100 µm.