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

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.

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

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica) to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti). The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms), but is absent in the early blood meal and final stages (procyclic and metacyclic forms). Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required.

Leishmania major glycosylation mutants require phosphoglycans (lpg2-) but not lipophosphoglycan (lpg1-) for survival in permissive sand fly vectors.

BACKGROUND: Sand fly species able to support the survival of the protozoan parasite Leishmania have been classified as permissive or specific, based upon their ability to support a wide or limited range of strains and/or species. Studies of a limited number of fly/parasite species combinations have implicated parasite surface molecules in this process and here we provide further evidence in support of this proposal. We investigated the role of lipophosphoglycan (LPG) and other phosphoglycans (PGs) in sand fly survival, using Leishmania major mutants deficient in LPG (lpg1(-)), and the phosphoglycan (PG)-deficient mutant lpg2(-). The sand fly species used were the permissive species Phlebotomus perniciosus and P. argentipes, and the specific vector P. duboscqi, a species resistant to L. infantum development. PRINCIPAL FINDINGS: The lpg2(-) mutants did not survive well in any of the three sand fly species, suggesting that phosphoglycans and/or other LPG2-dependent molecules are required for parasite development. In vitro, all three L. major lines were equally resistant to proteolytic activity of bovine trypsin, suggesting that sand fly-specific hydrolytic proteases or other factors are the reason for the early lpg2(-) parasite killing. The lpg1(-) mutants developed late-stage infections in two permissive species, P. perniciosus and P. argentipes, where their infection rates and intensities of infections were comparable to the wild type (WT) parasites. In contrast, in P. duboscqi the lpg1(-) mutants developed significantly worse than the WT parasites. CONCLUSIONS: In combination with previous studies, the data establish clearly that LPG is not required for Leishmania survival in permissive species P. perniciosus and P. argentipes but plays an important role in the specific vector P. duboscqi. With regard to PGs other than LPG, the data prove the importance of LPG2-related molecules for survival of L. major in the three sand fly species tested.

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.