Stage-specific molecular and morphogenic markers were used to follow the kinetics of appearance, number, and position of metacyclic promastigotes developing during the course of L. major infection in a natural vector, Phlebotomus papatasi. Expression of surface lipophosphoglycan (LPG) on transformed promastigotes was delayed until day 3, and continued to be abundantly expressed by all promastigotes thereafter. An epitope associated with arabinose substitution of LPG side-chain oligosaccharides was detected on the surface of a low proportion of midgut promastigotes beginning on day five, and on up to 60% of promastigotes on days 10 and 15. In contrast, 100% of the parasites egested from the mouthparts during forced feeding of 15-day infected flies stained strongly for this epitope. A metacyclic-associated transcript (MAT-1) was used in in situ hybridization studies to demonstrate the positioning of metacyclics in the anterior gut. L. major mutants defective in the expression of side-chain sugars on LPG, which are involved in midgut adhesion, were obtained by negative selection using lectins and antibodies. The absence of specific sidechain sugars was shown by in vitro biosynthetic studies using the phosphoglycan backbone as acceptor to be due to the lack of a galactosyl transferase. Attempts to clone the transferase by functional complimentation of the defective gene have been frustrated by the high frequency of chromosomal integration of the cosmid in the rescued clones. The cosmid DNA has recently been recovered by restriction digests of total DNA from the rescued phenotypes, re-ligation into the cosmid vector for bacterial transformation and cloning. The importance of the sand fly peritrophic matrix to the transformation, growth and development of Leishmania major in P. papatasi was investigated. Sand flies were infected by membrane feeding on amastigotes containing whole blood, with or without added chitinase. Transformation to and growth of promastigotes were observed in dissected midguts from all control flies by 36 hrs, whereas no viable promastigotes were observed in midguts from the chitinase-treated flies. When chitinase-treated flies were infected with logarithmic phase promastigotes instead of amastigotes, viable midgut infections developed. These data suggest an essential role for the peritrophic matrix in protecting the parasite during its early stage of development from the potentially lethal activities of the bloodfed midgut.
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