Binding of Leishmania infantum Lipophosphoglycan to the Midgut Is Not Sufficient To Define Vector Competence in Lutzomyia longipalpis Sand Flies.

Iliano V Coutinho-Abreu,Shaden Kamhawi,Rodrigo P Soares,Jesus G Valenzuela,Timothy R Wilson,Waldionê De Castro,Albert Descoteaux,Valéria M Borges,James Oristian,Claudio Meneses,Ira J Blader

doi:10.1128/msphere.00594-20

Abstract

The major surface lipophosphoglycan (LPG) of Leishmania parasites is critical to vector competence in restrictive sand fly vectors in mediating Leishmania attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of Leishmania remains elusive. We tested binding of Leishmania infantum wild-type (WT), LPG-defective (Δlpg1 mutants), and add-back (Δlpg1 + LPG1) lines to sand fly midguts in vitro and their survival in Lutzomyia longipalpis sand flies in vivoLe. infantum WT parasites attached to the Lu. longipalpis midgut in vitro, with late-stage parasites binding to midguts in significantly higher numbers than were seen with early-stage promastigotes. Δlpg1 mutants did not bind to Lu. longipalpis midguts, and this was rescued in the Δlpg1 + LPG1 lines, indicating that midgut binding is mediated by LPG. When Lu. longipalpis sand flies were infected with the Le. infantum WT or Le. infantum Δlpg1 or Le. infantum Δlpg1 + LPG1 line of the BH46 or BA262 strains, the BH46 Δlpg1 mutant, but not the BA262 Δlpg1 mutant, survived and grew to numbers similar to those seen with the WT and Δlpg1 + LPG1 lines. Exposure of BH46 and BA262 Δlpg1 mutants to blood-engorged midgut extracts led to mortality of the BA262 Δlpg1 but not the BH46 Δlpg1 parasites. These findings suggest that Le. infantum LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, but that it is not necessary to prevent Le. infantum evacuation along with the feces in the permissive vector Lu. longipalpisIMPORTANCE It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to Leishmania parasites. However, the permissiveness of other sand flies with respect to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector, Lutzomyia longipalpis We found that LPG-mediated midgut binding persists in late-stage parasites. This observation is of relevance for the understanding of vector-parasite molecular interactions and suggests that only a subset of infective metacyclic-stage parasites (metacyclics) lose their ability to attach to the midgut, with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in Leishmania infantum retention in the midgut of Lutzomyia longipalpis, a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be a complex issue and should be investigated on a strain-specific basis.

Highlights

IMPORTANCE It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to Leishmania parasites
4-day-old parasites did not bind intact, unopened, Lu. longipalpis midguts, pointing to the specificity of parasite binding to the sand fly midgut epithelium (Fig. 1C)
Incubation of P. papatasi midguts with purified PGs from procyclic Le. major parasites prevented the binding of procyclic Le. major parasites [6], and Le. major Δlpg1 parasites cannot develop in P. papatasi or P. duboscqi [25, 26] sand flies

Summary

Introduction

IMPORTANCE It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to Leishmania parasites. Leishmania relies on breakdown of the PM, mediated by a sand fly-secreted chitinase, to escape its confinement [2] After this step, the parasites attach to the midgut epithelium [6, 11], and such attachment in restrictive vectors requires specific carbohydrate side chains on the surface of the parasite that bind a specific receptor on the midgut microvilli [6, 12]. It has been demonstrated that variations in the nature of the side chain sugars decorating the LPG molecule of nonmetacyclic stages (nonmetacyclics) confer specificity to interactions with vectors [16] These side chains are modified to ensure release of metacyclic parasites (metacyclics) from the midgut [16]; in some instances, the LPG molecule conformationally prevents binding of sugars to the midgut [16, 19,20,21]. The permissive sand fly vectors, such as Phlebotomus perniciosus, Phlebotomus argentipes, and Lutzomyia longipalpis, are capable of supporting multiple Leishmania species [22,23,24]

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Conclusion