Chitin biosynthesis and the mosquito peritrophic matrix

Mosquito-borne pathogens initiate their infections in the vector by being ingested with a blood meal, and consequently the first barrier they face in establishing an infection is the midgut. Pathogens spending more than a few hours within the midgut must transverse the peritrophic matrix and midgut epithelium to reach specific tissues required for development and/or transmission. Therefore, the peritrophic matrix serves as the first physical barrier that must be circumvented. Chemical composition of the peritrophic matrix involves the presence of chitin, proteins and glycoproteins. Little is known about chitin biosynthesis, despite chitin's critical role in insects. The purpose of this work was to characterize the biochemical pathway and its regulatory mechanisms required for chitin biosynthesis in the midgut of Aedes aegypti, elucidate the role of chitin in peritrophic matrix formation, and to determine the biological function of the peritrophic matrix. cDNAs of all five enzymes in the chitin biosynthetic pathway were cloned and the transcriptional response of each gene to blood-feeding was characterized. Among the five genes, transcripts of AeGfat and AeCs, the first and last genes in the pathway, are up-regulated after blood-feeding, suggesting these two genes play regulatory roles in chitin biosynthesis at the transcriptional level. At the post-translational level, recombinant AeGFAT activity is sensitive to feedback inhibition by UDP-GlcNAc. Furthermore, when expression of either AeGfat or AeCs is knocked-down using RNAi, formation of the peritrophic matrix is disrupted, indicating that chitin is synthesized de novo in response to blood-feeding and is an essential component of the peritrophic matrix. Then, by disrupting peritrophic matrix formation by RNAi, biological functions of the peritrophic matrix were investigated. The results showed the presence or absence of a peritrophic matrix has no effect on the development of Brugia pahangi. However, infectivity of Plasmodium gallinaceum is lower, as measured by oocyst intensity, when the peritrophic matrix is absent. The peritrophic matrix also seems to localize proteolytic enzymes along the periphery of the blood bolus during the first 24 hr after blood feeding. Finally, it was demonstrated that the absence of the peritrophic matrix does not influence mosquito fecundity or longevity.