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Division of Infectious Diseases, Department of Medicine, University of CaliforniaSan Diego School of Medicine, La Jolla
Indirect evidence has suggested the existence of a second chitinase gene, PgCHT2, in the avian malaria parasite Plasmodium gallinaceum. We have now identified PgCHT2 as the orthologue of the P. falciparum chitinase gene PfCHT1, a malaria transmissionblocking target. Computational phylogenetic evidence and biochemical and cell biological functional data support the hypothesis that an avian-related Plasmodium species was the ancestor of both P. falciparum and P. reichenowi, and this single lineage gave rise to another lineage of malaria parasites, including P. vivax, P. knowlesi, P. berghei, P. yoelii, and P. chabaudi. A recombinant PfCHT1/PgCHT2-neutralizing single-chain antibody significantly reduced P. falciparum and P. gallinaceum parasite transmission to mosquitoes. This single-chain antibody is the first antiP. falciparum effector molecule to be validated for making a malaria transmissionrefractory transgenic Anopheles species mosquito. P. gallinaceum is a relevant animal model that facilitates a mechanistic understanding of P. falciparum invasion of the mosquito midgut.
Malaria is the most important parasitic disease of humans: hundreds of millions of people are infected annually, resulting in enormous morbidity and an estimated 13 million deaths. Malaria transmission begins when a mosquito injects infectious sporozoites into a vertebrate host while ingesting a blood meal, and it continues when a mosquito ingests a gametocyte-containing blood meal. A complex developmental process ensues in the mosquito midgut after ingestion of a blood meal. Male and female gametes merge to form zygotes that elongate into the invasive motile form, the ookinete. The ookinete must traverse the chitin-containing peritrophic matrix surrounding the ingested blood meal en route to invading the midgut epithelium to become a sporozoite-forming oocyst [1]. The ookinete secretes a family 18 chitinase [24] that facilitates parasite penetration and traversal of the peritrophic matrix, as has been shown in gene knockout studies [5, 6]; membrane feeding assays with allosamidin, which is a family 18 chitinase inhibitor [7]; and membrane feeding assays with chitinase-specific antibodies [8, 9]. A monoclonal antibody (MAb) raised against the recombinant P. falciparum chitinase PfCHT1, designated 1C3, neutralizes PfCHT1 enzymatic activity, recognizes a presumptive non-PgCHT1 chitinase in the avian malaria parasite P. gallinaceum, and significantly reduces the infectivity of both P. falciparum and P. gallinaceum in mosquitoes [8, 9].
The P. gallinaceum transmissionblocking effect of 1C3, the delineation of a novel secretion-associated structure in the apical end of the P. gallinaceum ookinete, and Western immunoblotting [8] suggested that a second PfCHT1-related chitinase gene, provisionally designated PgCHT2, existed in the avian malaria parasite [3, 8, 10]. Given the malaria transmissionblocking properties of 1C3 and its recognition of both PfCHT1 in P. falciparum ookinetes and PgCHT2 in P. gallinaceum ookinetes, we performed the present study to determine whether PgCHT2 encodes the orthologue of PfCHT1, and, if so, whether a recombinant single-chain antibody derived from the 1C3 hybridoma would reduce P. falciparum infectivity in Anopheles species mosquitoes and P. gallinaceum infectivity in Aedes aegypti mosquitoes. The characteristics of PgCHT2 and the presence of 2 chitinase genes in P. gallinaceum suggest that an avian malaria parasite was the ancestor of both rodent and primate lineages of Plasmodium species and gave rise to the chitinase of P. falciparum and the closely related chimpanzee malaria parasite P. reichenowi. The recognition of both PfCHT1 and PgCHT2 by the same recombinant single-chain antibody validates P. gallinaceum as a model system for understanding the mechanistic role that these chitinases play in ookinete invasion of the mosquito midgut. Furthermore, these data provide evidence of a new effector gene that has potential utility for the generation of malaria transmissionrefractory transgenic mosquitoes and provides insight into the mechanism of anti-chitinase malaria transmissionblocking antibodies.
MATERIALS AND METHODS
Parasites, mosquitoes, and cell lines.
P. gallinaceum strain 8a was maintained by cyclical passage through chickens and Aedes aegypti mosquitoes. P. falciparum strain 3D7 was maintained in vitro in continuous cultivation. The antiPfCHT1 MAbproducing hybridoma clone 1C3 has been described elsewhere [4]. The animal experimentation was approved by the University of CaliforniaSan Diego Institutional Animal Care and Use Committee and followed US federal guidelines.
Assembly and recombinant expression of the 1C3 ScFv gene.
mRNA was extracted from 5 × 106 1C3 hybridoma cells using the QuickPrep mRNA Purification Kit (Amersham Pharmacia Biotech). By use of the Mouse ScFv Module/Recombinant Phage Antibody System (Amersham Pharmacia Biotech), the first-strand cDNA was synthesized from 1C3 mRNA by reverse transcription using random hexamer priming. The 1C3 heavy variable (VH) and light variable (VL) segments were separately amplified from template cDNA using the following primers: 1C3-ScFv5-CATGCATGCCTATGGCCCAGGTGAAACTGCAG and 1C3-ScFv3-TCCCCGCGGCCGTTTTATTTCCAACTTTGTCC. The reaction conditions were 30 cycles at 94°C for 30 s, 50°C for 30 s, and 68°C for 30 s. The VH and VL products were gel-purified, quantified, and assembled into a single gene containing a DNA linker fragment by use of a 2-step polymerase chain reaction (PCR) protocol. The first reaction was 7 cycles at 94°C for 1 min, 63°C for 4 min, and 72°C for 1 min; the second reaction was 30 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min. The product of the full-length ScFv gene synthesis reaction was purified with a blunt-ended microspin column (10 mmol/L ATP, 10 mmol/L dNTP, 10 U of T4 DNA polymerase, and 10 U of T4 kinase), was cloned into pUC18, and was electroporated into Escherichia coli DH10B cells. The identity of recombinant ScFv genecontaining colonies was confirmed by SfiI and NotI restriction analysis and sequencing. For expression, the 1C3 ScFv gene was digested with the restriction enzymes SphI and SacII and was cloned into the insect cell expression vector pMIB/V5-HisB (Invitrogen), which carries the N-terminal honeybee melittin secretion signal for directing extracellular secretion of recombinant fusion proteins and a C-terminal peptide containing the V5 epitope and the His6 tag for detection and purification of the recombinant proteins, respectively.
High Five insect cells were transfected with 2 g of pMIB/V5-HisB-1C3-ScFv using 9 L of CellFECTIN Reagent (Invitrogen). Transfected cells were seeded into a 6-well tissue culture plate in 2 mL of Ultimate Insect serum-free medium and were incubated at 27°C. The insect cells and the medium were harvested at a density of 3 × 106 cells/mL. Cell lysates and culture medium were tested for 1C3 ScFv expression by Western immunoblot analysis, and anti-V5 epitope MAb (Invitrogen) was used for detection. Clonal cell lines were selected with Blasticidin S at a concentration of 50 g/mL and were maintained at a concentration of 10 g/mL. The pMIB/V5-HisB vector constitutively expresses protein under the control of the transactivating OpOE2 promoter of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus [11]. Recombinant 1C3 ScFv was purified with nickel-affinity chromatography and was analyzed by 4%20% SDS-PAGE. Its identity was confirmed by Edman degradation amino-terminal sequencing.
Immunofluorescence microscopy.
Parasites were fixed on glass slides with 100% acetone for 20 min at -20°C and were rehydrated by immersion of slides in 2 changes of 1× PBS at room temperature for 5 min/change. For membrane permeabilization and blocking of nonspecific binding, fixed cells were incubated in PBS/3% bovine serum albumin/3% Triton X-100 at room temperature for 1 h. The preparations were then incubated with recombinant 1C3 ScFv (5 g/mL) at room temperature for 1 h and then were incubated with fluorescein isothiocyanate (FITC)labeled antiV5 epitope MAb (1 : 200 dilution; Invitrogen) in PBS/3% bovine serum albumin/3% Triton X-100. Slides were mounted with Gelvatol (Molecular Probes). Microscopic examination was performed with a DeltaVision deconvolution immunofluorescence microscope system (Applied Precision).
Binding of recombinant 1C3 ScFv to P. falciparum chitinase PfCHT1.
Soluble, enzymatically active, E. coliproduced, recombinant PfCHT1 was expressed as described elsewhere [3]. Ninety-sixwell Immunolon 2 microplates (Dynex) were coated with 4 g/mL purified recombinant PfCHT1 in 100 mmol/L sodium carbonate buffer overnight at 4°C, were washed with PBS/0.05% Tween 20, were blocked with PBS/0.05% Tween 20/5% nonfat dry milk, and then were incubated with recombinant 1C3 ScFv, 1C3, and negative control isotype antibody for 3 h at 37°C. Binding was detected using horseradish peroxidaselabeled antiV5 epitope MAb and 3,3,5,5-tetramethylbenzidine (Sigma catalog no. T8865), and the optical density at 450 nm was measured.
Neutralization of recombinant PgCHT2 and PfCHT1 enzymatic activity by recombinant 1C3 ScFv.
Soluble, enzymatically active, E. coliproduced, recombinant PgCHT2 was expressed in E. coli strain Rosetta-gami 2 by cloning the postsecretory signal peptide open-reading frame of PgCHT2 into the expression vector pET32b (Novagen). Recombinant PgCHT2 was purified with nickel-affinity chromatography, and elution of protein was performed with 300 mmol/L imidazole. Recombinant PgCHT2 or PfCHT1 was added at a concentration of 1 g/L to individual wells of 96-well black plates to yield linearly increasing enzymatic activity over 30 min, as determined by a kinetic microfluorimetric assay. Recombinant 1C3 ScFv, 1C3, and an IgG1 MAb of irrelevant specificity (against dengue virus and used as an isotype negative control) were added to each well (final volume, 200 L), and the mixture was incubated for 30 min. Chitinase substrate (10 L of 125 mol/L 4-methylumbelliferyl-N,N,N--D-triacetylchitotrioside; Sigma) was then added, and the enzyme reaction was monitored by kinetic fluorescence detection (Dynatek Fluorolite 1000; excitation, 365 nm; emission, 450 nm).
Experimental feeding of mosquitoes to assess malaria transmissionblocking properties of antibodies.
To assess the effect that recombinant 1C3 ScFv has on P. falciparum infectivity in mosquitoes, in vitrocultivated P. falciparum strain 3D7 gametocytes (a mixture of day 14 and day 17 gametocytes) were used in membrane feeding assays to colonized Anopheles gambiae strain G3 and Anopheles stephensi mosquitoes. Membrane feeding assays of P. falciparum were performed using glass membrane feeders.
To assess the effect that recombinant 1C3 ScFv has on P. gallinaceum infectivity in Aedes aegypti mosquitoes, 40 female Aedes aegypti mosquitoes, aged 5 or 7 days postemergence, were placed in individual cartons, were starved for 24 h, and then were fed on infectious blood combined with the various antibodies for 20 min. The preparation was maintained at 37°C in water-jacketed membrane feeders. After 15 min of feeding, unengorged mosquitoes were removed. The remaining mosquitoes were maintained with 10% sucrose at 26°C and 70%80% relative humidity until dissection of midguts 7 days after the feeding. Data were analyzed by the Mann-Whitney U test.
RESULTS
Identification and analysis of the P. gallinaceum and P. reichenowi orthologues of the P. falciparum chitinase PfCHT1.
Indirect evidence has suggested the existence of a second chitinase gene in the avian malaria parasite P. gallinaceum [3, 8, 10]. By use of the amino-acid sequence of PfCHT1 in a BLAST search of the newly completed P. gallinaceum 3× coverage genome database, a sequence of a second chitinase gene, PgCHT2, was found. Independent sequencing of a cDNA clone obtained from P. gallinaceum ookinetes confirmed the genomic and predicted amino-acid sequence of PgCHT2 (figure 1). Similarly, the complete genomic sequence of the PfCHT1 orthologue, PrCHT1, was found in the P. reichenowi genome database. Analysis of the predicted primary amino-acid sequences of PgCHT2 and PrCHT1 confirmed that these 2 genes were orthologues of PfCHT1 (figures 1 and 2). All 3 are short forms of Plasmodium species chitinase, which lack both proenzyme and putative chitin-binding domains found in the long form of the chitinase (figures 1 and 2A), have a shared signal peptide length of 24 aarather than of 20 aa, as in all long forms of chitinaseand have a tyrosine rather than a tryptophan adjacent to the perfectly conserved proton-donating glutamic acid in the active site of the enzyme (figure 1). Furthermore, the epitope that was recognized by 1C3 and was previously mapped to recombinant PfCHT1 is present in both PgCHT2 and PrCHT1 (figure 1) but is absent in the long forms of the chitinase in other Plasmodium species [8]. An unrooted phylogenetic tree of the conserved catalytic region further supports the clustering of PgCHT2, PfCHT1, and PrCHT1 at a distance from the other chitinases (figure 2B). This analysis also showed that PgCHT1, the other chitinase of P. gallinaceum, is more closely related to the primate malaria parasite chitinases PvCHT1 and PkCHT1 than to the 3 rodent malaria parasite chitinases (figure 1B). The predicted catalytic domains of PfCHT1 and PgCHT2 have 72% amino-acid identity, which is greater identity than the predicted catalytic domains of PfCHT1 and PgCHT2, whose amino-acid sequences were only 37% identical.
Construction and expression of the single-chain antibody IC3 gene recognizing P. falciparum PfCHT1.
The cDNAs encoding the VH and VL segments of the P. falciparum chitinase PfCHT1neutralizing 1C3 were separately amplified by reverse-transcription PCR from mRNA obtained from cloned 1C3 hybridoma cells. The VH and VL cDNAs were synthesized into a single gene using a DNA linker encoding (Gly4Ser)3. The synthetic gene was cloned into the insect cell expression vector pMIB/V5-HisB, which is fused at its 5 end to the honey bee mellitin secretory signal sequence and at its 3 end to the V5 epitope followed by a His6 tag (figure 3A). This construct was verified by sequencing.
The pMIB/V5-HisB-1C3-ScFv plasmid was used to transform High Five insect cells and stable transformants that were selected by Blasticidin S drug pressure and cloned. A stably transfected clonal line constitutively secreted recombinant 1C3 ScFv into the culture medium (figure 3B). Affinity-purified recombinant 1C3 ScFv, which was purified to apparent homogeneity as assessed by Coomassie blue staining of SDS-PAGEseparated protein (figure 3C), was used in additional experiments to assess binding to recombinant PfCHT1 and the effect on P. gallinaceum and P. falciparum infectivity in mosquitoes.
Indirect immunofluorescence detection of recombinant 1C3 ScFv binding to PgCHT2 in P. gallinaceum ookinetes.
Deconvolution indirect immunofluorescence assay (IFA) microscopy was performed to determine whether recombinant 1C3 ScFv was bound to PgCHT2 expressed by mature P. gallinaceum ookinetes. Binding of recombinant 1C3 ScFv was detected with an FITC-labeled antiV5 epitope MAb. As was observed with the parental MAb 1C3, recombinant 1C3 ScFv was localized to an apical structure in the ookinete (figure 4B). In the negative control experiments, the FITC-labeled anti-V5 epitope MAb did not significantly bind to ookinetes in the absence of recombinant 1C3 ScFv (figure 4A), which indicates that the indirect IFA was specific for PgCHT2, the target of the single-chain antibody.
Recombinant 1C3 ScFv binding to and neutralizing of recombinant P. falciparum chitinase PfCHT1.
The binding of affinity-purified recombinant 1C3 ScFv was determined by ELISA with E. coliproduced recombinant PfCHT1, the antigen against which the parental MAb 1C3 was raised. Recombinant 1C3 ScFv was produced as a fusion protein with the V5 epitope, to allow for specific detection. Recombinant 1C3 ScFv bound specifically to recombinant PfCHT1, as detected with an antiV5 epitope MAb (figure 5A); binding of an isotype control MAb and the parental MAb 1C3 were indistinguishable from background levels of binding.
The ability of recombinant 1C3 ScFv to neutralize recombinant PfCHT1 and PgCHT2 chitinase activity was determined using a kinetic microfluorimetric assay that measured the release of free 4-methylumbelliferone from 4-methylumbelliferyl-N,N,N--D-triacetylchitotrioside [3]. The parental MAb 1C3 has been shown elsewhere to inhibit PfCHT1 enzymatic activity [8]. Recombinant 1C3 ScFv neutralized the enzymatic activity of both PfCHT1 (figure 5B) and PgCHT2 (figure 5C) in a dose-dependent manner.
Determination of malaria transmissionblocking activity of recombinant 1C3 ScFv.
The ability of recombinant 1C3 ScFv to inhibit sporogonic development of P. falciparum and P. gallinaceum in mosquitoes was assessed by membrane feeding assays. Previous data have established that the parental MAb 1C3 significantly reduced P. falciparum infectivity in Anopheles gambiae and Anopheles stephensi mosquitoes and P. gallinaceum infectivity in Aedes aegypti mosquitoes [8, 9]. 1C3 and recombinant 1C3 ScFV reproducibly and significantly reduced the number of oocysts and the proportion of infected mosquitoes when in vitrocultivated P. falciparum gametocytes were fed to either Anopheles gambiae or Anopheles stephensi mosquitoes (table 1). 1C3 and recombinant 1C3 ScFv also reproducibly and significantly reduced the number of P. gallinaceum oocysts and the proportion of infected Aedes aegypti mosquitoes (table 2).
DISCUSSION
We have demonstrated that the chitinase of the human malaria parasite P. falciparum and the chimpanzee malaria parasite P. reichenowi are the orthologues of a newly identified chitinase of the avian malaria parasite P. gallinaceum. P. gallinaceum is the only Plasmodium species that has been demonstrated, to date, to have 2 chitinase genes. An insect cellproduced recombinant single-chain antibody recognized, bound to, and neutralized the activity of both the P. falciparum chitinase PfCHT1 and the P. gallinaceum chitinase PgCHT2 and reduced the infectivity of both P. falciparum and P. gallinaceum in mosquitoes. These datataken together with previous demonstrations that 1C3, an anti-PfCHT1 MAb, has transmission-reducing activity for both P. falciparum [9] and P. gallinaceum [8]provide further support for the relevance of the P. gallinaceumAedes aegypti model for understanding the biochemical mechanisms of how chitinase allows the P. falciparum ookinete to invade the mosquito midgut.
The data presented here validate the first effector molecule against P. falciparum with the potential for creating a malaria transmissionrefractory transgenic Anopheles species mosquito. Other anti-Plasmodium effector molecules, including single-chain antibodies, the SM1 peptide, and a honeybee phospholipase, have been validated in animal models of malaria transmission. These molecules have been demonstrated to reduce the infectivity of P. berghei in Anopheles stephensi mosquitoes [1214], and a virus-expressed single-chain antibody reduced P. gallinaceum sporozoite infectivity in Aedes aegypti mosquito salivary glands [15]. To our knowledge, this is the first validation of an effector molecule for creating a P. falciparumrefractory transgenic mosquito. An important consideration in future experiments with recombinant 1C3 ScFv or any other P. falciparum transmissionblocking molecule is whether such interventions would have to be completely effective to justify their testing and deployment. Mathematical modeling of malaria transmission dynamics suggests that even partial reduction of oocyst counts in mosquitoes is likely have a substantial impact on malaria transmission dynamics [16, 17]. These observations demonstrate that the P. gallinaceum model of transmission in Aedes aegypti mosquitoes is directly relevant to studies of P. falciparum transmission in Anopheles species mosquitoes, particularly at the level of the peritrophic matrix, and have future relevance to the development of malaria transmissionblocking strategies.
Previous phylogenetic analyses of Plasmodium species using ribosomal RNA gene sequences [1820] have strongly suggested that P. falciparum and the closely related P. reichnowi share an ancestor with avian malaria parasites. However, these analyses did not functionally relate the evolutionary relationship of the phylogenetic lineages of the Plasmodium genus to either a vertebrate or a mosquito host. The present analysis of the phylogenetic relationships between chitinase genes of the different clades of the Plasmodium genus provides specific insight into parasite-vector interactions. The ookinete-secreted chitinase is only known to function in parasite penetration of the peritrophic matrix within the midgut of mosquitoes. Because the mosquito is the definitive host of Plasmodium species, our observations suggest that an important speciation event occurred at the parasite-mosquito interface. The most parsimonious explanation for the relationship between the chitinases of P. falciparum and P. reichenowi, on the one hand, and the chitinases of P. vivax, P. knowlesi, and the rodent malaria parasites, on the other hand, is that an avian-type malaria parasite, or an ancestor that gave rise to avian-type malaria parasites, was the ancestor of both. During the process of adaptation to new mosquito vectors, the long chitinase (in P. falciparum and P. reichenowi) or the short chitinase (in the other parasites) was lost, which resulted in only 1 chitinase gene in either lineage. The functional significance of the different chitinase forms vis à vis penetration of the peritrophic matrices of different mosquito species remains to be understood at the biochemical level, although data have been published that suggest that varying tropisms in Plasmodium species for different species and genera of mosquitoes is not likely due simply to the parasite's ability to penetrate any specific type of peritrophic matrix [21].
The demonstration of the transmission-blocking properties of the single-chain antibody 1C3 for both P. falciparum and P. gallinaceum has 2 major implications. First, the 1C3 ScFv gene may be an additional useful effector molecule for creating a malaria transmissionrefractory transgenic mosquito. The malaria transmissionblocking activity of either recombinant 1C3 ScFv or the parental MAb 1C3 was not absolute when the protein was added to a blood meal. However, if recombinant 1C3 ScFv were to be continuously secreted into the midgut after ingestion of an infectious blood meal and concentrated at the blood meal/peritrophic matrix interface immediately adjacent to the epithelial lining, its malaria transmissionblocking effect would likely be longer lasting and more complete than when the antibody (either in the form of a MAb or a recombinant ScFv) is added once to an experimental infectious blood meal, because under those circumstances it is subject to proteolytic degradation. Second, that the 1C3 MAb and ScFv cross-react with both the PfCHT1 and the PgCHT2 chitinase and have transmission-blocking effects supports the functional relevance of the P. gallinaceum model for transmission-blocking studies of P. falciparum, particularly at the level of how ookinetes interact with the peritrophic matrix. It is unlikely that recombinant 1C3 ScFv interferes with the activity of PgCHT1, the other P. gallinaceum chitinase, because we have shown that the parental MAb 1C3 does not react with this protein, as determined by Western immunoblot, and PgCHT1 lacks the 1C3-recognized epitope [8], so that the effect of 1C3 is specific for PfCHT1/PgCHT2. It is unlikely that recombinant 1C3 ScFv would have binding specificity, particularly a new specificity for PgCHT1, that the parental MAb does not have.
The demonstration that recombinant 1C3 ScFV has chitinase-binding and chitinase-neutralizing activity provides insight into potential mechanisms of its malaria transmissionblocking effects. Given the monovalency and the lack of an intact Fc component of single-chain antibodies, neither surface molecule cross-linking nor complement-mediated lysis is likely to be responsible for the malaria transmissionblocking properties of recombinant 1C3 ScFv. Therefore, the most likely mechanism by which recombinant 1C3 ScFv blocks parasite transmission is neutralization of secreted [3] or cell surfaceassociated [8] chitinase enzymatic activity. Given the orthologous relationship between PgCHT2 and PfCHT1, these mechanistic insights from the P. gallinaceum model system are likely applicable to P. falciparum.
The experimental results described here provide additional justification that the ookinete-expressed chitinase of Plasmodium species is a malaria transmissionblocking target and validate a gene encoding a P. falciparum chitinaseneutralizing single-chain antibody as an effector gene that could be used for creating a P. falciparumrefractory transgenic mosquito. Targeting sequential molecular processes of parasite infection of the mosquito midgut, before oocyst formation when parasite numbers are exponentially amplified, may be synergistic in the development of a malaria parasiteresistant transgenic mosquito. Therefore, the strategy of targeting a relatively late event in midgut invasionookinete penetration of the peritrophic matrix mediated by chitinasewill be complementary to the targeting of the zygote/ookinete surface molecule Pfs25 and ookinete mechanisms of binding to and penetrating the mosquito midgut epithelial cell [22].
Acknowledgments
We thank Biovest International; the US National Cell Culture Center, for manufacturing hollow fiberproduced, protein Apurified monoclonal antibody 1C3 (supported by US Public Health Service grant 5U42RR005991); and James Feramisco, director of the In Vivo and Cellular Imaging Shared Resource at the University of CaliforniaSan Diego (supported by US Public Health service grant 5P01HL066941), for assistance in imaging.
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