Bee Venom Phospholipase Inhibits Malaria Parasite Development in Transgenic Mosquitoes

Abstract

Malaria kills millions of people every year, and new control measures are urgently needed. The recent demonstration that (effector) genes can be introduced into the mosquito germ line to diminish their ability to transmit the malaria parasite offers new hope toward the fight of the disease (Ito, J., Ghosh, A., Moreira, L. A., Wimmer, E. A. & Jacobs-Lorena, M. (2002)Nature, 417, 452–455). Because of the high selection pressure that an effector gene imposes on the parasite population, development of resistant strains is likely to occur. In search of additional antiparasitic effector genes, we have generated transgenicAnopheles stephensi mosquitoes that express the bee venom phospholipase A2 (PLA2) gene from the gut-specific and blood-inducible Anopheles gambiaecarboxypeptidase (AgCP) promoter. Northern blot analysis indicated that the PLA2 mRNA is specifically expressed in the guts of transgenic mosquitoes with peak expression at ∼4 h after blood ingestion. Western blot and immunofluorescence analyses detected PLA2 protein in the midgut epithelia of transgenic mosquitoes from 8 to 24 h after a blood meal. Importantly, transgene expression reducedPlasmodium berghei oocyst formation by 87% on average and greatly impaired transmission of the parasite to naive mice. The results indicate that PLA2 may be used as an additional effector gene to block the development of the malaria parasite in mosquitoes. Malaria kills millions of people every year, and new control measures are urgently needed. The recent demonstration that (effector) genes can be introduced into the mosquito germ line to diminish their ability to transmit the malaria parasite offers new hope toward the fight of the disease (Ito, J., Ghosh, A., Moreira, L. A., Wimmer, E. A. & Jacobs-Lorena, M. (2002)Nature, 417, 452–455). Because of the high selection pressure that an effector gene imposes on the parasite population, development of resistant strains is likely to occur. In search of additional antiparasitic effector genes, we have generated transgenicAnopheles stephensi mosquitoes that express the bee venom phospholipase A2 (PLA2) gene from the gut-specific and blood-inducible Anopheles gambiaecarboxypeptidase (AgCP) promoter. Northern blot analysis indicated that the PLA2 mRNA is specifically expressed in the guts of transgenic mosquitoes with peak expression at ∼4 h after blood ingestion. Western blot and immunofluorescence analyses detected PLA2 protein in the midgut epithelia of transgenic mosquitoes from 8 to 24 h after a blood meal. Importantly, transgene expression reducedPlasmodium berghei oocyst formation by 87% on average and greatly impaired transmission of the parasite to naive mice. The results indicate that PLA2 may be used as an additional effector gene to block the development of the malaria parasite in mosquitoes. Worldwide mortality due to malaria has increased in the past decade mainly because of parasite and mosquito resistance to drugs and insecticides, respectively, and the lack of effective vaccines (1Webster D. J. Public Health Policy. 2001; 22: 23-33Google Scholar). Genetically engineering mosquito vectors for refractoriness to malaria parasites is a strategy for reducing disease transmission that should be explored. Plasmodium, the causative agent of malaria, has to complete a complex developmental program in the mosquito for transmission to occur. The first interactions between the parasite and the mosquito occur in the midgut lumen, where the parasite has to traverse two barriers, the peritrophic matrix, and the midgut epithelium (2Shahabuddin M. Cociancich S. Zieler H. Parasitol. Today. 1998; 14: 493-497Google Scholar, 3Ghosh A. Edwards M.J. Jacobs-Lorena M. Parasitol. Today. 2000; 16: 196-201Google Scholar). Because the gut is a closed compartment that limits diffusion, antimalarial compounds secreted into the midgut lumen are expected to efficiently target the initial stages of parasite development. Previous studies have demonstrated that venom phospholipases A2 (PLA2s) 1The abbreviations used are: PLA2, phospholipase A2; AgCP, A. gambiae carboxypeptidase; UTR, untranslated region; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; EGFP, enhanced GFP; DAPI, 4′,6-diamidino-2-phenylindole. strongly inhibit oocyst formation when administered to mosquitoes with an infectious blood meal (4Zieler H. Keister D.B. Dvorak J.A. Ribeiro J.M.C. J. Exp. Biol. 2001; 204: 4157-4167Google Scholar). Although the mechanism of inhibition has not been established, it is known that PLA2 does not kill ookinetes and does not interfere with their development in vitro. Furthermore, inhibition of oocyst formation did not depend on PLA2 enzymatic activity. It is possible that PLA2 inhibits ookinete invasion by modifying the properties of the midgut epithelial membranes that are invaded by the parasite. The best candidates for driving the expression of foreign gene products to be secreted into the mosquito midgut are the promoters of bloodmeal-inducible midgut genes because of their strength, tissue specificity, and synchrony of expression with parasite ingestion by the mosquito. In this context, we have shown that the Anopheles gambiae and Aedes aegypti carboxypeptidase promoters can be used to drive strong expression of recombinant protein in the midgut of transgenic mosquitoes (5Moreira L.A. Edwards M.J. Adhami F. Jasinskiene N. James A.A. Jacobs-Lorena M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10895-10898Google Scholar) and also that they can drive the expression of a parasite blocking peptide (6Ghosh A. Ribolla P.E. Jacobs-Lorena M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13278-13281Google Scholar) in transgenic mosquitoes (7Ito J. Ghosh A. Moreira L.A. Wimmer E.A. Jacobs-Lorena M. Nature. 2002; 417: 452-455Google Scholar). Although the latter results indicate that genetic manipulation of mosquito vectors is a promising strategy for reducing malaria transmission, it is also important to consider that the use of a single effector gene is likely to lead to rapid selection of resistant parasites. Thus, we have searched for additional effector genes whose products interfere with parasite development by mechanisms different from the previously developed ones (6Ghosh A. Ribolla P.E. Jacobs-Lorena M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13278-13281Google Scholar, 7Ito J. Ghosh A. Moreira L.A. Wimmer E.A. Jacobs-Lorena M. Nature. 2002; 417: 452-455Google Scholar). The results reported in this article suggest that bee venom PLA2 may be used as an alternate effector gene. The full-length cDNA of the honeybee phospholipase A2 gene was cloned for the first time by relying on the previously published partial sequence (GenBankTM accession numberX16709). Messenger RNA was isolated from 105 venom glands dissected out of newly emerged honeybees using the Micro Fast-Track mRNA isolation kit from Invitrogen. The mRNA was reverse-transcribed with Superscript II reverse transcriptase (Invitrogen) using the SMART cDNA synthesis kit (Clontech) in the presence of a primer specific to the previously published 3′ end of the honeybee PLA2 cDNA (5′-ccgccgtctagataaataaacgatctcgaagtggtactc-3′), as well as a custom-synthesized SMART primer (5′-agcagtggtttaaacgcagagtggccattatggccggg-3′). The first strand cDNA was PCR-amplified using the same two primers and Platinum-Taq high fidelity polymerase (Invitrogen), and the PCR product was cloned with the TOPO-TA cloning kit (Invitrogen). The resulting construct BV PLA2 5-7consisted of the full-length bee venom PLA2 open reading frame flanked by 5′- and 3′-untranslated sequences inserted into the pCR-TOPO vector (Invitrogen). The PLA2 sequence thus obtained included the entire signal peptide, which was only partially present in previously published sequences, as well as a portion of the 5′-untranslated region (UTR). Inserts from several identical clones were sequenced, and the sequence was used to prepare PCR primers for the construction of the mosquito expression constructs. A 3.9-kb fragment from theA. gambiae carboxypeptidase gene (8Edwards M.J. Lemos F.J.A. Donnely-Doman M. Jacobs-Lorena M. Insect Biochem. Mol. Biol. 1997; 27: 1063-1072Google Scholar) (AgCP5′; containing the promoter, 5′-UTR, and signal peptide) was amplified by PCR from a pBluescript AgCPgenomic subclone using AgCP Kpn(5′-GGTACCCTCGGCCGCTTCGACACT-3′) and T7(5′-GTAATACGACTCACTATAGGGC-3′) primers and cloned into pGemT-easy. Bee venom PLA2 coding region (450 bp) was cloned into pGemT-easy (Promega) from a cDNA clone using primersPLA2K(5′-GGTACCTGGCAAATCAGGGAT-3′) and PLA2B(5′-GGATCCTTATCAATACTTGCGAAGATC-3′). The AgCP5′in pGemT-easy was digested with KpnI, and the resulting 1.8-kb fragment was ligated into the PLA2 plasmid (KpnI-digested). AgCP3′(555 bp) (untranslated 3′ region) was obtained by PCR amplification with primersAgCP3BH(5′-GGATCCTGAAGTCTCTCCTACCGG-3′) or AgCP3SC(5′-CCGCGGTAAGGCTAGCATTGCCA-3′) on an AgCP pBluescript genomic subclone and cloned into pGemT-easy. Both theAgCP/PLA2 and AgCP in pGemT were digested with BamHI and SacII and the AgCP3′-UTR fragment ligated to AgCP5/PLA2(pGemT-easy). A NotI fragment containingAgCP/PLA2/AgCP3was cloned intopSLfa1180faplasmid (9Horn C. Wimmer E.A. Dev. Genes Evol. 2000; 210: 630-637Google Scholar) that contains unique FseI and AscI sites. The plasmid was then cut with these enzymes and cloned into piggyBac[3xP3-EGFPafm]plasmid. The final transposon plasmid pBac[3xP3-EGFP(AgCPPLA2)]contains 1.7 kb of AgCP promoter, around 100 bp ofAgCP coding region to provide the signal sequence, 0.45 kb of PLA2 coding region, and 0.58 kb of AgCP-3′ UTRapart from the EGFP sequence (see Fig. 1). Anopheles stephensi embryos were injected as described (7Ito J. Ghosh A. Moreira L.A. Wimmer E.A. Jacobs-Lorena M. Nature. 2002; 417: 452-455Google Scholar, 10Catteruccia F. Nolan T. Loukeris T.G. Blass C. Savakis C. Kafatos F.C. Crisanti A. Nature. 2000; 405: 959-962Google Scholar). Embryos were injected with a mixture of the transposon construct (0.5 mg/ml) and of the piggyBac helper plasmid (11Handler A.M. Harrell R.A. Insect Mol. Biol. 1999; 8: 449-457Google Scholar) (0.3 mg/ml), both purified on Qiagen midi-prep columns. No heat shock was performed. The surviving adults were pooled into families as follows. Adult injected males (three to five G0 mosquitoes) were crossed with 5–10 virgin non-injected females. Between three and six G1virgin injected females were pooled and mated with three to five non-injected males. Females were allowed to feed on blood, and G1 embryos were collected. Transformants were selected by screening cold-immobilized larvae for EGFP expression using a dissecting fluorescence microscope at a wavelength of 490 nm. Individual G1 transgenic mosquitoes (males or females) from positive families were mated with non-transgenic mosquitoes. Progenies of these crosses were checked for integration of the transgene as follows. Total genomic DNA was isolated from transgenic and wild type mosquitoes as described (12Black I.V. Black W.C. Munstermann L.E. Beaty B. Marquardt W.C. The Biology of Disease Vectors. University Press of Colorado, Niwot, Colorado1996: 438-470Google Scholar) but with 100 μg/ml proteinase K treatment at 52 °C for 3 h and followed by phenol:chloroform extractions and precipitation. DNA digested withBglII was separated on a 0.8% agarose gel, blotted, and hybridized with [α-32P]dCTP-labeled piggyBacprobe originating from the piggyBac vector left arm (∼0.8-kb SalI fragment from pBac[3xP3-EGFP(AgCPPLA2)](see Fig. 1, probe a)). Female mosquito guts and carcasses (whole body minus gut) and male guts were dissected from 4–5-day old adults. All tissues were immediately frozen in an ethanol/dry ice bath and stored at −80 °C. To determine the temporal profile ofPLA2 expression in transgenic mosquitoes, guts were dissected at increasing time intervals following a blood meal. Total RNA was extracted with TRI-Reagent (Molecular Research Center). Around 2.5 μg of total RNA was separated in each lane of a 1.5% agarose-formaldehyde gel and blotted by capillary action to a nylon membrane (Gene Screen). Hybridization was performed first with aPLA2 probe (450 bp of coding region amplified by PCR; see Fig. 1, probe b) and then with a mitochondrial rRNA probe (13Lemos F.J.A. Cornel A.J. Jacobs-Lorena M. Insect Biochem. Mol. Biol. 1996; 26: 651-658Google Scholar) as a loading control. Guts were dissected in phosphate-buffered saline (PBS) at different times after a blood meal. The equivalent of 0.4 guts/lane was analyzed by electrophoresis on a 15% polyacrylamide/SDS gel followed by electrotransfer to a polyvinylidene fluoride membrane (Millipore). A prestained protein ladder (Benchmark, Invitrogen) was loaded on the same gel. The membrane was incubated with an anti-rabbit bee venom phospholipase A2 polyclonal antibody (Accurate; 1:2,000 dilution), and the bound antibody was detected with an anti-rabbit immunoglobulin, horseradish peroxidase-linked (New England Biolabs, 1:3,000 dilution) by exposing the blots to x-ray films. About 2 × 107 Plasmodium berghei parasites (ANKA 2.34) were naive and were used to feed a of non-transgenic and transgenic mosquitoes. that were separated after 24 h and at °C with were and the of was and non-transgenic mosquitoes were in the same and allowed to feed on a single that were separated after 24 h and at °C with mosquitoes were separated into and each mosquito was allowed to feed on a single naive mosquitoes were glands were dissected and in a of PBS, and were with a The of each was followed by blood on and Female mosquito guts were dissected at different times after a blood meal in and to a The gut were with a to the as described (6Ghosh A. Ribolla P.E. Jacobs-Lorena M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13278-13281Google in at six to times with PBS, and for h with at °C. The guts were then incubated at °C with an anti-rabbit bee venom phospholipase A2 antibody (Accurate; 1:2,000 The were times and incubated in the for 2 h with a fluorescein anti-rabbit The gut were with a (Molecular and by fluorescence Guts from mosquitoes were dissected in in PBS, for 2 h at and three times in in a the guts were with and in and were The were in at followed by in a To were and in a The were in and in for 2 h at and incubated at with an venom phospholipase A2 antibody (Accurate; in blocking were three times for with blocking and incubated for 2 h at with anti-rabbit were by with with was by fluorescence at was on the same by and with the using the expression of the A. stephensi we the that of the promoter, the 5′-UTR, and the signal peptide from the (8Edwards M.J. Lemos F.J.A. Donnely-Doman M. Jacobs-Lorena M. Insect Biochem. Mol. Biol. 1997; 27: 1063-1072Google Scholar) to the coding sequence of the bee venom and the AgCP gene was inserted into which has unique for cloning into the piggyBac vector (9Horn C. Wimmer E.A. Dev. Genes Evol. 2000; 210: 630-637Google Scholar). vector screening of mosquitoes fluorescence can be detected in the of both larvae and mosquitoes. Importantly, the of the not interfere with of the protein (7Ito J. Ghosh A. Moreira L.A. Wimmer E.A. Jacobs-Lorena M. Nature. 2002; 417: 452-455Google Scholar). expression in only a tissues may be for mosquito piggyBac to be an vector for A. stephensi embryos adults were obtained and pooled into different transgenic mosquito and were from two families in the following The the surviving adults was were for A. stephensi with F. Nolan T. Loukeris T.G. Blass C. Savakis C. Kafatos F.C. Crisanti A. Nature. 2000; 405: 959-962Google Scholar) and for the following A. stephensi with piggyBac Crisanti A. F. J. Biol. 2002; A. aegypti with (5Moreira L.A. Edwards M.J. Adhami F. Jasinskiene N. James A.A. Jacobs-Lorena M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10895-10898Google Scholar, N. Cornel A.J. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1998; Scholar, Insect Mol. Biol. 2000; aegypti with (5Moreira L.A. Edwards M.J. Adhami F. Jasinskiene N. James A.A. Jacobs-Lorena M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10895-10898Google Scholar, Jasinskiene N. L. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1998; Scholar). To that the transgene was and to determine the of DNA from each mosquito line was analyzed by blot genomic DNA was digested with blotted, and hybridized with a probe from the piggyBac left arm 1, probe shown in Fig. each mosquito line a single transgene at a different that integration in each of the families that and from different No signal was detected with DNA from mosquitoes not of the mRNA was by Northern analysis A single of the expected was detected M. M.J. J. Biochem. Scholar). The mRNA was present in the midgut of mosquitoes and was strongly by blood ingestion at ∼4 with the of expression of the A. gambiaecarboxypeptidase gene (8Edwards M.J. Lemos F.J.A. Donnely-Doman M. Jacobs-Lorena M. Insect Biochem. Mol. Biol. 1997; 27: 1063-1072Google Scholar). expression at h was also but the for this are not mRNA expression as well as tissue and indicate that the AgCP sequence contains the analysis that the antibody detected a protein that was in a blood-inducible in the of transgenic mosquitoes The of the protein on was identical to that of an high pressure bee venom PLA2 protein not gut equivalent of protein lane was for PLA2 Importantly, the recombinant protein a peak of expression between 8 and 24 h after a blood with the time of ookinete invasion of the midgut A. Edwards M.J. Jacobs-Lorena M. Parasitol. Today. 2000; 16: 196-201Google Scholar). the protein was not which with the Northern detected the protein in gut and in gut In the signal in epithelia from guts h after a blood in a signal was detected at 24 by 24 the of the PLA2 been secreted into the lumen and was of the gut PLA2 can be as a signal between the epithelium and the blood meal in transgenic gut and this with the Western of midgut guts from non-transgenic or from mosquitoes, dissected at different times after a blood meal to the left of were and The were incubated with a to bee venom phospholipase A2 followed by a anti-rabbit antibody The of the midgut epithelial are with The blood meal is also To the of recombinant PLA2 expression berghei we both transgenic and non-transgenic mosquitoes on the same and the of that in each of mosquitoes. indicated in and oocyst formation were strongly in transgenic mosquitoes. In five oocyst formation was from to inhibition of PLA2 expression on berghei oocyst of mosquitoes in of midgut and oocyst gut in transgenic oocyst gut in control mosquitoes) × of oocyst was for as by the and non-transgenic mosquitoes were on the transgenic and non-transgenic mosquitoes and were separated and after gut and the of gut was of mosquitoes in of midgut and 100 oocyst gut in transgenic oocyst gut in control mosquitoes) × of oocyst was for as by the in a new and non-transgenic mosquitoes were on the transgenic and non-transgenic mosquitoes and were separated and after gut and the of gut was The of recombinant gene expression on the ability of mosquitoes to transmit the parasite to was by single mosquitoes feed on naive and these reported in 2 and 3 of in every transgenic non-transgenic mosquitoes and the of in glands of transgenic mosquitoes was the ability of transgenic mosquitoes to transmit the parasite to naive was strongly In three out of transmission of berghei parasites from transgenic mosquitoes to naive was and in a the of transgenic mosquitoes that the parasite was in control wild type mosquitoes the that inhibition of parasite development was a of the of a mosquito gene transposon or of The following strongly these the same of parasite development and was in different mosquito in which the transposon at different of the mosquito 2 and and the same was when PLA2 was to wild type mosquitoes (4Zieler H. Keister D.B. Dvorak J.A. Ribeiro J.M.C. J. Exp. Biol. 2001; 204: 4157-4167Google and berghei developed well and in transgenic mosquitoes that express only and resistance gene F. Nolan T. Loukeris T.G. Blass C. Savakis C. Kafatos F.C. Crisanti A. Nature. 2000; 405: 959-962Google Scholar) as in wild type mosquitoes. T. and F. The PLA2 protein secreted in the midgut lumen of transgenic mosquitoes is likely for inhibition of ookinete midgut as with the administered protein (4Zieler H. Keister D.B. Dvorak J.A. Ribeiro J.M.C. J. Exp. Biol. 2001; 204: 4157-4167Google of transgenic mosquitoes is mosquitoes is a from only an of the were The of was in transgenic in non-transgenic mosquitoes in as by the each control non-transgenic and transgenic mosquitoes were on the same To transmission, single mosquitoes were on naive after the infectious blood meal. The of each mosquito was dissected immediately after on the and the of was reported in 2 and The of each was by a of blood on alternate that parasites by were to be reported in is a from only an of the were The of was in transgenic in non-transgenic mosquitoes in as by the in a new each control non-transgenic and transgenic mosquitoes were on the same To transmission, single mosquitoes were on naive after the infectious blood meal. The of each mosquito was dissected immediately after on the and the of was reported in 2 and The of each was by a of blood on alternate that parasites by were to be reported in The of phospholipases to their as and membrane is of their enzymatic M. Sci. 1999; Scholar). it has been shown that PLA2 development when enzymatic was (4Zieler H. Keister D.B. Dvorak J.A. Ribeiro J.M.C. J. Exp. Biol. 2001; 204: 4157-4167Google that PLA2 to membrane PLA2 on and formation and did not ookinete on that this does not kill the parasite (4Zieler H. Keister D.B. Dvorak J.A. Ribeiro J.M.C. J. Exp. Biol. 2001; 204: 4157-4167Google Scholar). and the of the the that PLA2 may be by with the interactions between Plasmodium and the midgut mosquitoes were in and as as the an of the blood meal was 24 h after of may also be A of the of transgenic as with wild type mosquitoes is In these that expression of the gene in the midgut of transgenic mosquitoes their ability to Plasmodium development and to transmit the parasite to this PLA2 also with development and transmission in A. gambiae (4Zieler H. Keister D.B. Dvorak J.A. Ribeiro J.M.C. J. Exp. Biol. 2001; 204: 4157-4167Google we that PLA2 be effective in transmission in this important of to interfere transmission by expression of A. Jasinskiene N. James A.A. A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: Scholar) or single M. J. E. James A.A. J. 2000; Scholar) in A. aegypti have their in transgenic mosquitoes to be from this that expression of a midgut and peptide, also inhibits development and transmission of the parasite (6Ghosh A. Ribolla P.E. Jacobs-Lorena M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13278-13281Google J. Ghosh A. Moreira L.A. Wimmer E.A. Jacobs-Lorena M. Nature. 2002; 417: 452-455Google Scholar). The of to development is for of the transgenic mosquito to malaria transmission in the is because the Plasmodium is known for and the of the of resistant parasite strains to be at can consider to be that to be of wild mosquito an of the ability of to the that the parasites resistance to the foreign effector gene and The reported strongly suggest that genetic of mosquito is and a toward the of containing the of for we have to on a that may insecticides, and mosquito vectors a of effector for two containing and honeybees and also and for with the of mosquitoes and mice. are also to of for and