The role of surface glycoconjugates in Leishmania midgut attachment examined by competitive binding assays and experimental development in sand flies

SUMMARY Binding of promastigotes to the sand fly midgut epithelium is regarded as an essential part of the Leishmania life cycle in the vector. Among Leishmania surface molecules putatively involved in attachment to the sand fly midgut, two GPI-anchored molecules are the most prominent: lipophosphoglycan (LPG) and promastigote surface protease gp63. In this work, we examined midgut attachment of Leishmania lines mutated in GPI-anchored molecules and compared results from 2 different techniques: in vivo development in sand flies and in vitro competitive binding assays using fluorescently labelled parasites. In combination with previous studies, our data provide additional support for (1) an LPG-independent parasite-binding mechanism of Leishmania major within the midgut of the permissive vector Phlebotomus perniciosus, and provide strong support for (2) the crucial role of L. major LPG in specific vector Phlebotomus papatasi, and (3) a role for Leishmania amazonensis gp63 in Lutzomyia longipalpis midgut binding. Moreover, our results suggest a critical role for GPI-anchored proteins and gp63 in Leishmania mexicana attachment to L. longipalpis midguts, as the wild type (WT) line accounted for over 99% of bound parasites

Comparative binding of enriched parasite populations to <i>Lu. longipalpis</i> midguts.

<p>All populations were compared to the binding of the nectomonad-enriched culture. (A) <i>L. mexicana</i> (11 experiments) (B) <i>L. infantum</i> (5 experiments). Cultures were enriched for: procyclic promastigotes (P); nectomonad promastigotes (N); leptomonad promastigotes (L) and metacyclic promastigotes (M). Each bar represents the mean percentage ± S.D. and the asterisks indicate a significant difference to the nectomonad population (>20 dissected guts examined per experiment).</p

Binding of two <i>Leishmania</i> populations to a sand fly midgut.

<p>In the example shown, <i>L. mexicana</i> nectomonad promastigotes are labelled with Syto Blue and <i>L. mexicana</i> leptomonads with Syto Green, and are bound to a flattened <i>Lu. longipalpis</i> midgut. The gut was photographed after washing, and all the bound promastigotes were alive as revealed by movements of their cell bodies. Some areas with fewer promastigotes can be seen, but these had no obvious distribution.</p

Inhibitor of cysteine peptidase does not influence the development of Leishmania mexicana in Lutzomyia longipalpis

It has been proposed that the natural cysteine peptidase inhibitor ICP of Leishmania mexicana protects the protozoan parasite from insect host proteolytic enzymes, thereby promoting survival. To test this hypothesis, L. mexicana mutants deficient in ICP were evaluated for their ability to develop in the sand fly Lutzomyia longipalpis. No significant differences were found between the wild-type parasites, two independently derived ICP-deficient mutants, or mutants overexpressing ICP; all lines developed similarly in the sand fly midgut and produced heavy late-stage infections. In addition, recombinant L. mexicana ICP did not inhibit peptidase activity of the midgut extracts in vitro. We conclude that ICP has no major role in promoting survival of L. mexicana in the vectorial part of its life cycle in L. longipalpis

Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay

The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s) that mediate binding is not fully understood