Two serine proteases from Anopheles dirus haemocytes exhibit changes in transcript abundance after infection of an incompatible rodent malaria parasite, Plasmodium yoelii.
ABSTRACT Serine proteases are involved in regulation of innate immune responses, such as haemolymph coagulation, melanization reaction and antimicrobial peptide synthesis. Although several serine proteases have been characterized in Anopheles gambiae (A. gambiae), few were cloned from other malaria vectors. In this study, we identified three cDNA fragments of serine proteases (AdSp1, AdSp2 and AdSp3) from haemocytes of an oriental malaria vector, Anopheles dirus (A. dirus), by cloning of fragments amplified with degenerate primers into the T-vector. RT-PCR analysis demonstrated that both AdSp1 and AdSp3 genes were also expressed in salivary gland. Basic local alignment search tool (BLAST) search found that both AdSp1 and AdSp3 were highly similar in sequence to A. gambiae Sp14A and Sp14D2, insects prophenoloxidase activating enzyme (PPAE) and Drosophila protease easter. Semi-quantitative RT-PCR indicated the transcription level of both AdSp1 and AdSp3 in haemocytes of A. dirus infected with Plasmodium yoelii (P. yoelii) was significant higher than that fed on 5% glucose or normal mouse blood at 7 days after the infectious meal (p<0.05), when P. yoelii oocysts began to be melanized by A. dirus. Our results indicated that both AdSp1 and AdSp3 might play an important role during melanotic encapsulation of P. yoelii by A. dirus.
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ABSTRACT: The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.Science 11/1986; 234(4776):607-10. · 31.03 Impact Factor
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ABSTRACT: Analogous to blood coagulation and complement activation in mammals, some insect defense responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in hemolymph. We recently isolated Manduca sexta serpin-6 from hemolymph of the bacteria-challenged larvae, which selectively inhibited proPO-activating proteinase-3 (PAP-3) (Wang, Y., and Jiang, H. (2004) Insect Biochem. Mol. Biol. 34, 387-395). To further characterize its structure and function, we cloned serpin-6 from an induced fat body cDNA library using a PCR-derived probe. M. sexta serpin-6 is 55% similar in amino acid sequence to Drosophila melanogaster serpin-5, an immune-responsive protein. We produced serpin-6 in an Escherichia coli expression system and purified the soluble protein by nickel affinity and hydrophobic interaction chromatography. The recombinant protein specifically inhibited PAP-3 and blocked proPO activation in vitro in a concentration-dependent manner. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that the cleavage site of serpin-6 is between Arg373 and Ser374. Serpin-6 is constitutively present in hemolymph of naive larvae, and its mRNA and protein levels significantly increase after a bacterial injection. The association rate constant of serpin-6 and PAP-3 is 2.6 x 10(4) m(-1) s(-1), indicating that serpin-6 may contribute to the inhibitory regulation of PAP-3 in the hemolymph. We also identified the covalent complex of serpin-6 and PAP-3 in induced hemolymph by immunoaffinity chromatography and mass spectrometry. Furthermore, immulectin-2, serine proteinase homologs, proPO, PO, attacin-2, and a complex of serpin-6 and hemolymph proteinase-8 were also detected in the proteins eluted from the immunoaffinity column using serpin-6 antibody. These results suggest that serpin-6 plays important roles in the regulation of immune proteinases in the hemolymph.Journal of Biological Chemistry 05/2005; 280(14):14341-8. · 4.65 Impact Factor
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ABSTRACT: Cell lines from the malaria vector Anopheles gambiae have been established as a tool for the study of the mosquito innate immune system in vitro. Here, we describe the first continuous insect cell line that produces prophenoloxidase (PPO). This cell line (4a-3B) expresses constitutively six PPO genes, three of which are novel (PPO4, PPO5, and PPO6). The PPO genes show distinct temporal expression profiles in the intact mosquito, spanning stages from the embryo to the adult in an overlapping manner. Transient induction of larva-specific PPO genes in blood-fed adult females suggests that the developmental hormone 20-hydroxyecdysone may be involved in PPO gene regulation. Indeed, exposure of 4a-3B cells to 20-hydroxyecdysone in culture results in induction of those PPO genes that are mainly expressed in early developmental stages, and repression of PPO5, which is preferentially expressed at the adult stage. The cell line shows bacteria-induced immune transcripts that encode defensin and Gram-negative bacteria-binding protein, but no induction of PPO transcripts. This cell line most likely derives from a hemocyte lineage, and represents an appropriate in vitro model for the study of the humoral and cellular immune defenses of A. gambiae.Journal of Biological Chemistry 05/1999; 274(17):11727-35. · 4.65 Impact Factor