Abstract
BackgroundHematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.Methodology/Principal FindingsHz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.Conclusions/SignificanceHerein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.
Highlights
During the course of evolution, hematophagous organisms have developed and selected an array of strategies to counteract heme cytotoxicity to adapt successfully to blood feeding [1]
Hz was first described in Plasmodium falciparum [7], but over the last decade Hz has been described in a number of other blood-sucking organisms such as the helminth worms, Schistosoma mansoni [8] and Echinostoma trivalvis [9], and the bird-infecting protozoan, Hemoproteus columbae [10]
Effect of a-glucosidase inhibitors on Hz formation Erythritol (100 mM) and castanospermine (30 mM), specific inhibitors of a-glucosidase, Diethypyrocarbonate (DEPC) (10 mM), which react with and modify histidine residues, and a polyclonal antibody raised against a-glucosidase from D. peruvianus (1:2500) were used in order to investigate the correlation between the a-glucosidase and Hz formation activities (Figure 1A and 1B)
Summary
During the course of evolution, hematophagous organisms have developed and selected an array of strategies to counteract heme cytotoxicity to adapt successfully to blood feeding [1]. R. prolixus is a hematophagous hemipteran that sequesters hemoglobin-derived heme into a dark-brown pigment named Hz which is an insoluble and less reactive substance [2]. This is the first line of defense against heme toxicity in the midgut of this insect [6]. Hz was first described in Plasmodium falciparum [7], but over the last decade Hz has been described in a number of other blood-sucking organisms such as the helminth worms, Schistosoma mansoni [8] and Echinostoma trivalvis [9], and the bird-infecting protozoan, Hemoproteus columbae [10] These discoveries have broadened the interest into understanding the mechanism of Hz formation. The involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated
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