RESUMEN
In addition to modulating blood meal digestion and protecting the midgut epithelial cells from mechanical and chemical damage, a biological function attributed to the mosquito type I peritrophic matrix (PM) is preventing or reducing pathogen invasion, especially from Plasmodium spp. Previously, we demonstrated that chitin is an essential component of the PM and is synthesized de novo in response to blood feeding in Aedes aegypti. Therefore, knocking down chitin synthase expression by RNA interference severely disrupts formation of the PM. Utilizing this artificial manipulation, we determined that the absence of the PM has no effect on the development of Brugia pahangi or on the dissemination of dengue virus. However, infectivity of Plasmodium gallinaceum is lower, as measured by oocyst intensity, when the PM is absent. Our findings also suggest that the PM seems to localize proteolytic enzymes along the periphery of the blood bolus during the first 24 hours after blood feeding. Finally, the absence of the PM does not affect reproductive fitness, as measured by the number and viability of eggs oviposited.
Asunto(s)
Aedes/fisiología , Tracto Gastrointestinal/fisiología , Insectos Vectores , Animales , Brugia pahangi/fisiología , Pollos , Virus del Dengue/fisiología , Femenino , Gerbillinae , Interacciones Huésped-Patógeno , Oviposición , Plasmodium gallinaceum/fisiologíaRESUMEN
A previous report has shown that mosquito sterol carrier protein-2 inhibitors (SCPIs) are larvicidal to larvae of the yellowfever mosquito, Aedes aegypti (L.) (J. Lipid Res. 46: 650-657, 2005). In the current study, we tested SCPI-1 in an additional four mosquito species for larvicidal activities: Culex pipiens pipiens, Anopheles gambiae, Culex restuans, and Aedes vexans. Cholesterol accumulation in SCPI-treated Ae. aegypti fourth instars was examined. SCPI-1 is lethal to all tested mosquito species, with the LC50 value ranging from 5.2 to 15 microM when treatments started at the first to third instar. However, LC50 values increase to from 5.2 to 38.7 microM in treatments started at first and fourth instar, respectively. The results indicate that the lethal effect of SCPI-1 decreases with the growth of larvae, which suggests that SCPI-1 is more effective before the larvae reach final growth period (the last instar). SCPI-1 suppressed cholesterol uptake in Ae. aegypti fourth instars, suggesting that one of the modes of action of SCPI-1 is via reduction in cholesterol absorption.
Asunto(s)
Acetanilidas/antagonistas & inhibidores , Proteínas Portadoras/antagonistas & inhibidores , Culicidae/efectos de los fármacos , Insecticidas/farmacología , Quinolinas/antagonistas & inhibidores , Tiazoles/antagonistas & inhibidores , Acetanilidas/química , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Colesterol/metabolismo , Larva/efectos de los fármacos , Datos de Secuencia Molecular , Quinolinas/química , Tiazoles/químicaRESUMEN
Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 A resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [(3)H]cholesterol and [3H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Insectos/química , Animales , Transporte Biológico , Cristalografía por Rayos X , Culicidae , Ácidos Grasos/metabolismo , Femenino , Expresión Génica , Metabolismo de los Lípidos , Conformación Proteica , TritioRESUMEN
In mosquitoes, the peritrophic matrix is formed in response to blood feeding and can be a physical barrier when pathogens ingested with blood meal attempt to reach and transverse the midgut epithelium. The main components of the peritrophic matrix are chitin-biding-domain containing proteins, glycosylated proteins, and chitin fibrils. Chitin is synthesized from fructose-6-phosphate by a series of five enzymatic reactions. We previously found that blood feeding induces transcriptional up-regulation of glutamine: fructose-6-phosphate amidotransferase-1 (AeGfat-1) and chitin synthase (AeCs), the first and last enzymes of the biosynthetic pathway, respectively, in the midgut of Aedes aegypti. In this study, we demonstrated that formation of the peritrophic matrix is disrupted when the transcript abundance of either gene is knocked-down using RNAi methodologies. We also have shown that enzymatic activity of recombinant AeGFAT-1 is sensitive to feedback inhibition by UDP-N-acetylglucosamine, a substrate of chitin synthase. These findings demonstrate that in the midgut of adult Ae. aegypti, (1) chitin is synthesized de novo in response to blood feeding and is an essential component of the peritrophic matrix, and (2) chitin biosynthesis is negatively regulated, in part, by inhibitory sensitivity of AeGFAT-1 to UDP-N-acetylglucosamine.
Asunto(s)
Aedes/metabolismo , Quitina/metabolismo , Tracto Gastrointestinal/metabolismo , Animales , Sangre , Quitina/biosíntesis , Conducta Alimentaria , Fructosafosfatos/metabolismo , Tracto Gastrointestinal/anatomía & histología , Regulación de la Expresión Génica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Modelos Químicos , Regulación hacia ArribaRESUMEN
Mosquito midgut epithelial cells secrete digestive enzymes as well as components of the peritrophic matrix in response to blood-feeding. The peritrophic matrix is composed of proteins, glycoproteins and chitin fibrils in a proteoglycan matrix and may function to protect the midgut epithelium from mechanical damage and insult from pathogens and toxins. Chitin biosynthesis takes place via the hexosamine pathway converting fructose-6-phosphate to UDP-N-acetylglucosamine, which is then polymerized to chitin by chitin synthase. Glucosamine-6-phosphate N-acetyltransferase (GNA) is one of the hexosamine pathway enzymes and catalyzes the transfer of the acetyl group from acetyl-CoA to the primary amine of glucosamine-6-phosphate. We cloned and sequenced the GNA cDNA, gene (AeGna) and its putative promoter regions from Aedes aegypti. AeGna consists of five exons and four introns and lacks a TATA box near the transcription start site. The AeGna cDNA is 1.3 kb in length and the predicted protein is approximately 23.6 kDa. The amino acid sequence of AeGna has high homology to its orthologues. AeGna mRNA is constitutively expressed in all developmental stages and blood-feeding causes no obvious effect on levels of AeGna transcript in the midgut. The Km value of recombinant GNA for glucosamine-6-phosphate was 330 microM and the Km for acetyl-CoA was 500 microM.
Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/genética , Aedes/enzimología , Acetiltransferasas/metabolismo , Aedes/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/química , Glucosamina 6-Fosfato N-Acetiltransferasa , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A mosquito sterol carrier protein-2, AeSCP-2, has been shown to aid in the uptake of cholesterol in mosquito cells. The discovery of chemical inhibitors of AeSCP-2 is reported here. AeSCP-2 inhibitors (SCPIs) belong to several chemotypes of hydrophobic compounds. Those inhibitors competed with cholesterol for AeSCP-2, binding with relatively high binding affinities. In cultured insect cells, SCPIs reduced cholesterol uptake by as much as 30% at 1-5 microM concentrations. SCPIs were potent larvicides to the yellow fever mosquito, Aedes aegypti, and to the tobacco hornworm, Manduca sexta, with 50% lethal doses (LD50s) of 5-21 microM and 0.013-15 ng/mg diet, respectively. The results indicate that sterol carrier protein-2 has functional similarity in two different insect species.