Targeting essential Eimeria ninakohlyakimovae sporozoite ligands for caprine host endothelial cell invasion with a phage display peptide library.

Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe diarrhoea in young animals. Specific molecules that mediate E. ninakohlyakimovae host interactions and molecular mechanisms involved in the pathogenesis are still unknown. Although strong circumstantial evidence indicates that E. ninakohlyakimovae sporozoite interactions with caprine endothelial host cells (ECs) are specific, hardly any information is available about the interacting molecules that confer host cell specificity. In this study, we describe a novel method to identify surface proteins of caprine umbilical vein endothelial cells (CUVEC) using a phage display library. After several panning rounds, we identified a number of peptides that specifically bind to the surface of CUVEC. Importantly, caprine endothelial cell peptide 2 (PCEC2) and PCEC5 selectively reduced the infection rate by E. ninakohlyakimovae sporozoites. These preliminary data give new insight for the molecular identification of ligands involved in the interaction between E. ninakohlyakimovae sporozoites and host ECs. Further studies using this phage approach might be useful to identify new potential target molecules for the development of anti-coccidial drugs or even new vaccine strategies.

The Anopheles gambiae adult midgut peritrophic matrix proteome.

Malaria is a devastating disease. For transmission to occur, Plasmodium, the causative agent of malaria, must complete a complex developmental cycle in its mosquito vector. Thus, the mosquito is a potential target for disease control. Plasmodium ookinetes, which develop within the mosquito midgut, must first cross the midgut's peritrophic matrix (PM), a thick extracellular sheath that completely surrounds the blood meal. The PM poses a partial, natural barrier against parasite invasion of the midgut and it is speculated that modifications to the PM may lead to a complete barrier to infection. However, such strategies require thorough characterization of the structure of the PM. Here, we describe for the first time, the complete PM proteome of the main malaria vector, Anopheles gambiae. Altogether, 209 proteins were identified by mass spectrometry. Among them were nine new chitin-binding peritrophic matrix proteins, expanding the list from three to twelve peritrophins. Lastly, we provide a model for the putative interactions among the proteins identified in this study.

Expression of a mutated phospholipase A2 in transgenic Aedes fluviatilis mosquitoes impacts Plasmodium gallinaceum development.

The genetic manipulation of mosquito vectors is an alternative strategy in the fight against malaria. It was previously shown that bee venom phospholipase A2 (PLA2) inhibits ookinete invasion of the mosquito midgut although mosquito fitness was reduced. To maintain the PLA2 blocking ability without compromising mosquito biology, we mutated the protein-coding sequence to inactivate the enzyme while maintaining the protein's structure. DNA encoding the mutated PLA2 (mPLA2) was placed downstream of a mosquito midgut-specific promoter (Anopheles gambiae peritrophin protein 1 promoter, AgPer1) and this construct used to transform Aedes fluviatilis mosquitoes. Four different transgenic lines were obtained and characterized and all lines significantly inhibited Plasmodium gallinaceum oocyst development (up to 68% fewer oocysts). No fitness cost was observed when this mosquito species expressed the mPLA2.

Driving midgut-specific expression and secretion of a foreign protein in transgenic mosquitoes with AgAper1 regulatory elements.

The Anopheles gambiae adult peritrophic matrix protein 1 (AgAper1) regulatory elements were used to drive the expression of phospholipase A2 (PLA2), a protein known to disrupt malaria parasite development in mosquitoes. These AgAper1 regulatory elements were sufficient to promote the accumulation of PLA2 in midgut epithelial cells before a blood meal and its release into the lumen upon blood ingestion. Plasmodium berghei oocyst formation was reduced by approximately 80% (74-91% range) in transgenic mosquitoes. Blood-seeking behaviour and survival of AgAper1-PLA2 transgenic mosquitoes were comparable to sibling wild-type mosquitoes, while fertility was substantially lower. Ultrastructural studies suggest that decreased fitness is a consequence of internal damage to midgut epithelial cells.

Storage and secretion of Ag-Aper14, a novel peritrophic matrix protein, and Ag-Muc1 from the mosquito Anopheles gambiae.

The gene Ag-Aper14 encodes a novel peritrophic matrix (or peritrophic membrane; PM) protein in the mosquito Anopheles gambiae. The Ag-Aper14 protein is merely 89 amino acids long and has a single putative chitin-binding domain. Prior to blood feeding, the Ag-Aper14 protein is stored in secretory vesicles next to the epithelial cell lumenal surface. Immunoelectron microscopy has revealed that Ag-Aper14 co-localizes to the same secretory vesicles as another PM protein, Ag-Aper1, indicating a common mode of regulated secretion. Conversely, Ag-Muc1, an epithelial cell-surface protein, does not co-localize to these secretory vesicles and is detected only on the cell surface. After blood feeding, Ag-Aper14 is secreted and incorporated into the PM that surrounds the ingested blood.

Gene expression in Plasmodium: from gametocytes to sporozoites.

Completion of the complex developmental program of Plasmodium in the mosquito is essential for parasite transmission, yet this part of its life cycle is still poorly understood. In recent years, considerable progress has been made in the identification and characterization of genes expressed during parasite development in the mosquito. This line of investigation was greatly facilitated by the availability of the genome sequence of several Plasmodium, and by the application of approaches such as proteomics, microarrays, gene disruption by homologous recombination (gene knockout) and by use of subtraction libraries. Here, we review what is presently known about genes expressed in gametocytes and during the Plasmodium life cycle in the mosquito.

Storage and secretion of the peritrophic matrix protein Ag-Aper1 and trypsin in the midgut of Anopheles gambiae.

The gene Ag-Aper1 encodes a peritrophic matrix (PM) protein from the mosquito Anopheles gambiae. Ag-Aper1 gene expression and protein localization in the mosquito midgut were studied during the course of a blood meal. Ag-Aper1 mRNA abundance does not change appreciably during the course of blood ingestion and digestion. Prior to a blood meal, the protein is stored in secretory vesicles of midgut epithelial cells. Moreover, Ag-Aper1 colocalizes to the same secretory vesicles as trypsin, indicating that these proteins use a common secretory pathway. Blood feeding triggers the secretion of vesicle contents into the midgut lumen, after which Ag-Aper1 is incorporated into the PM. Newly synthesized Ag-Aper1 protein was again detected within the midgut epithelial cells at 60 h after blood ingestion.

The peritrophic matrix limits the rate of digestion in adult Anopheles stephensi and Aedes aegypti mosquitoes.

The peritrophic matrix (PM) is a chitin-containing acellular sheath that surrounds the blood meal and separates the food bolus from the midgut epithelium. Intense molecular traffic through the PM occurs during digestion. Digestive enzymes secreted by the midgut epithelium must traverse the PM to reach their substrates in the food bolus, and digestion products must cross the PM in the opposite direction to be absorbed by the epithelial cells. Here we report that the PM limits the rate of digestion. PM disruption by two independent means (chitinase and anti-PM antibodies) consistently increases the rate of blood digestion. The significance of these results in relation to PM function is discussed.

Plasmodium-mosquito interactions, phage display libraries and transgenic mosquitoes impaired for malaria transmission.

Malaria continues to kill millions of people every year and new strategies to combat this disease are urgently needed. Recent advances in the study of the mosquito vector and its interactions with the malaria parasite suggest that it may be possible to genetically manipulate the mosquito in order to reduce its vectorial capacity. Here we review the advances made to date in four areas: (1) the introduction of foreign genes into the mosquito germ line; (2) the characterization of tissue-specific promoters; (3) the identification of gene products that block development of the parasite in the mosquito; and (4) the generation of transgenic mosquitoes impaired for malaria transmission. While initial results show great promise, the problem of how to spread the blocking genes through wild mosquito populations remains to be solved.

Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library.

Despite vast efforts and expenditures in the past few decades, malaria continues to kill millions of persons every year, and new approaches for disease control are urgently needed. To complete its life cycle in the mosquito, Plasmodium, the causative agent of malaria, has to traverse the epithelia of the midgut and salivary glands. Although strong circumstantial evidence indicates that parasite interactions with the two organs are specific, hardly any information is available about the interacting molecules. By use of a phage display library, we identified a 12-aa peptide--salivary gland and midgut peptide 1 (SM1)--that binds to the distal lobes of the salivary gland and to the luminal side of the midgut epithelium, but not to the midgut surface facing the hemolymph or to ovaries. The coincidence of the tissues with which parasites and the SM1 peptide interact suggested that the parasite and peptide recognize the same surface ligand. In support of this hypothesis, the SM1 peptide strongly inhibited Plasmodium invasion of salivary gland and midgut epithelia. These experiments suggest a new strategy for the genetic manipulation of mosquito vectorial capacity.

Developmental regulation of an instability element from the Drosophila fushi tarazu mRNA.

The Drosophila fushi tarazu (ftz) mRNA is one of the shortest-lived metazoan mRNAs, and its instability is crucial for proper development of the embryo. Previously, we identified two cis-acting elements that are required for ftz mRNA degradation, one within the 5' one-third and another in the 3'UTR of the message. Here we focus on the 3'UTR element termed FIE3 (ftz instability element in the 3'UTR). To investigate the developmental regulation of the FIE3-dependent degrading activity we measured the abundance of an FIE3-containing mRNA in ovaries, unfertilized eggs, and different larval and adult tissues. We found that FIE3-degrading activity is present at all developmental stages and tissues examined, except in the ovary. Activation of the FIE3-dependent mRNA decay is independent of fertilization because it could be triggered by egg activation. Finally, we provide evidence that mutation of conserved elements within FIE3 had no effect on mRNA instability.

Amino acid residues involved in substrate binding and catalysis in an insect digestive beta-glycosidase.

A beta-glycosidase (M(r) 50000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl beta-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pK(a1)=4.9 and pK(a2)=7.5). Effect of pH on carbodiimide inactivation indicates that the pK(a) 7.5 group is a carboxyl. k(cat) and K(m) values were obtained for different p-nitrophenyl beta-glycosides and K(i) values were determined for a range of alkyl beta-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature beta-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E(187) (proton donor) and E(399) (nucleophile); (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite.

The peritrophic matrix of hematophagous insects.

The peritrophic matrix (PM) is an extracellular envelope that lines the digestive tract of most insects. It is thought to play key roles in protecting insects from pathogens and facilitating digestion. Until recently, little information was available on the molecular composition of the PM. This review summarizes recent progress in the study of the PM from hematophagous insects, with emphasis on molecular and physiological aspects. Topics discussed include the presence of chitin and protein diversity in the PM, cloning and characterization of genes encoding PM proteins, PM permeability, and the role of the PM as a barrier for pathogens.

Functional mapping of destabilizing elements in the protein-coding region of the Drosophila fushi tarazu mRNA.

The instability of the fushi tarazu (ftz) mRNA is essential for the proper development of the Drosophila embryo. Previously, we identified a 201-nucleotide instability element (FIE3) in the 3' untranslated region (UTR) of the ftz mRNA. Here we report on the identification of two additional elements in the protein-coding region of the message: the 63-nucleotide-long FIE5-1 and the 69-nucleotide-long FIE5-2. The function of both elements was position-dependent; the same elements destabilized RNAs when present within the coding region but did not when embedded in the 3' UTR of the hybrid mRNAs. We conclude that ftz mRNA has three redundant instability elements, two in the protein-coding region and one in the 3' UTR. Although each instability element is sufficient to destabilize a heterologous mRNA, the destabilizing activity of the two 5'-elements depended on their position within the message.

Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae.

Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [Afanasiev et al. (1994) Exp Parasitol 79: 322-339; Afanasiev et al. (1999) Virology 257: 62-72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139-159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations (Afanasiev etal., 1999; Allen-Muira et al. (1999) Virology 257: 54-61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day-old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10-0.15 M inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression.

Robust gut-specific gene expression in transgenic Aedes aegypti mosquitoes.

Genetic modification of the vectorial capacity of mosquito vectors of human disease requires promoters capable of driving gene expression with appropriate tissue and stage specificity. We report on the characterization in transgenic Aedes aegypti of two mosquito gut-specific promoters. A 1.4-kb DNA fragment adjacent to the 5' end of the coding region of the Ae. aegypti carboxypeptidase (AeCP) gene and a corresponding 3.4-kb DNA fragment at the 5' end of the Anopheles gambiae carboxypeptidase (AgCP) gene were linked to a firefly luciferase reporter gene and introduced into the Ae. aegypti germ line by using Hermes and mariner (Mos1) transposons. Six independent transgenic lines were obtained with the AeCP construct and one with the AgCP construct. Luciferase mRNA and protein were abundantly expressed in the guts of transgenic mosquitoes in four of the six AeCP lines and in the AgCP line. Expression of the reporter gene was gut-specific and reached peak levels at about 24 h post-blood ingestion. The AeCP and AgCP promoters can be used to drive the expression of genes that hinder parasite development in the mosquito gut.

A gut-specific serine protease from the malaria vector Aanopheles gambiae is downregulated after blood ingestion.

A chymotrypsin-like serine protease gene (AgChyL) was cloned from the mosquito Anopheles gambiae by a polymerase chain reaction (PCR)-based subtractive cDNA cloning strategy. AgChyL messenger RNA (mRNA) is abundant in the adult female gut prior to, and for 8 h following, a blood meal. During the peak of digestion, from 12 to 24 h following a blood meal, AgChyL mRNA abundance decreased to barely detectable levels. AgChyL mRNA was abundant again by 48 h following a blood meal. Recombinant pro-AgChyL was expressed in Escherichia coli. The pro-enzyme can be activated by trypsin. Activated AgChyL cleaves the synthetic chymotrypsin substrate succinyl-L-Ala-Ala-Pro-Phe-nitroanilide, but not two other synthetic chymotrypsin substrates or synthetic trypsin and elastase substrates. The potential role of AgChyL in the coordination of An. gambiae digestion is discussed.

Permeability and disruption of the peritrophic matrix and caecal membrane from Aedes aegypti and Anopheles gambiae mosquito larvae.

In mosquito larvae, the peritrophic matrix (PM) separates the gut contents from the intestinal epithelium. This report describes a new in vivo assay for estimating PM permeability. The assay also allows for assessment of the permeability of the caecal membrane, a structure that separates each caecum from the gut lumen. Permeability was estimated by the appearance of fluorescently-labeled dextrans (size range 4,400 to 2 million Da) within the gastric caecae of mosquito larvae. While the intact peritrophic matrix was impermeable to 2 million Da dextran particles, it was permeable to dextran particles of 148 kDa and smaller. The caecal membrane appears to have considerably smaller pores, being permeable only to dextrans of 19.5 kDa and smaller. The assay was also used to devise a treatment that disrupts the PM sufficiently to allow the passage of virus-sized particles. Dithiothreitol and to a lesser extent, chitinase were effective in disrupting the PM. Cycloheximide had a small effect; Polyoxin D, Pronase and calcofluor did not alter the permeability to 2 million Da dextran particles. Disruption of the PM is discussed in the context of infecting mosquitoes with retroviral transformation vectors.

The journey of the malaria parasite in the mosquito: hopes for the new century.

In this review, Anil Ghosh, Marten Edwards and Marcelo Jacobs-Lorena follow the journey of the Plasmodium parasite in the mosquito vector. At each developmental step, they highlight some of the major unanswered questions currently challenging cell and molecular biologists. A more thorough understanding of Plasmodium-mosquito interactions might lead to the development of mosquitoes unable to support parasite development.

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti.

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.