Leishmania major Glycosylation Mutants Require Phosphoglycans (lpg2−) but Not Lipophosphoglycan (lpg1−) for Survival in Permissive Sand Fly Vectors

Phlebotomine sand flies are small blood-feeding insects, medically important as vectors of protozoan parasites of the genus Leishmania. Sand flies species can be divided roughly into two groups, termed specific or permissive, depending on their ability to support development of one or a few strains vs. a broad spectrum of these parasites. In this study, we explored the ability of two Leishmania major glycocalyx mutants to survive within these different types of vectors. The lpg1− mutant, which specifically lacks lipophosphoglycan (LPG), was able to survive normally in two permissive species, Phlebotomus argentipes and P. perniciosus, but was only able to survive within the specific species P. duboscqi for a limited time prior to dissolution of the peritrophic matrix. Consistent with its classification as a specific sand fly vector, P. duboscqi was not able to support development of L. infantum. The lpg2− L. major mutant, which is a broader mutant and lacks all phosphoglycans including LPG and proteophosphoglycans, was unable to survive in all the three vector species tested. This study extends the knowledge on the role of Leishmania major surface glycoconjugates to development in three important vector species and gives supporting evidence for the existence of an LPG-independent mechanism for survival in sand flies, as well as the importance of LPG2-dependent glycoconjugates in parasite survival

Development of <i>L. major</i> mutants in <i>P. duboscqi</i>.

<p><i>Phlebotomus duboscqi</i> females were infected with <i>Leishmania major</i> wild type (WT) or mutants lacking LPG (<i>lpg1</i>⁻) or all <i>LPG2</i>-dependent molecules (<i>lpg2</i>⁻). Day 2 - dissection before defecation (48hours post-infection), day 9 - dissection after defecation. Infections were classified into three cathegories: heavy (more than 1000 promastigotes per gut) - black bars, moderate (100–1000) - grey bars, light (1–100) - white bars. Numbers above the bars indicate the number of dissected females.</p

Development of <i>L. major</i> mutants in <i>P. argentipes</i>.

<p><i>Leishmania major</i> lines tested were the same as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000580#pntd-0000580-g001" target="_blank">Fig. 1</a>. Day 2 - dissection before defecation (48 hours post-infection); day 5 - dissection after defecation. The intensity of infections was evaluated as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000580#pntd-0000580-g001" target="_blank">Fig. 1</a>. Numbers above the bars indicate the number of dissected <i>P. argentipes</i> females.</p

Development of <i>L. major</i> mutants in <i>P. perniciosus</i>.

<p><i>Leishmania major</i> lines tested and evaluation of infections were the same as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000580#pntd-0000580-g001" target="_blank">Fig. 1</a>. Numbers above the bars indicate the number of dissected <i>P. perniciosus</i> females.</p

Western blot of <i>P. duboscqi</i> midgut proteins incubated with various lectins.

<p>Lectins concanavalin A (Con) and <i>Pisum sativum</i> agglutinin (PSA) specifically reacted with <i>P. duboscqi</i> midgut lysate. Reaction of lectins <i>Helix pomatia</i> agglutinin (HPA), <i>Ricinus communis</i> agglutinin (RCA) and Soybean agglutinin (SBA) were negative.</p