Leishmania (Viannia) braziliensis metacyclic promastigotes purified using Bauhinia purpurea lectin are complement resistant and highly infective for macrophages in vitro and hamsters in vivo

Abstract

In this study we characterised metacyclogenesis in axenic culture of Leishmania (Viannia) braziliensis, the causative agent of mucocutaneous leishmaniasis in the New World. Metacyclogenesis of other species of Leishmania has been shown by morphological changes as well as molecular modifications in the lipophosphoglycan, the major cell surface glycoconjugate of the promastigotes. In order to obtain metacyclic forms of L. braziliensis we tested a panel of different lectins. Our results showed that Bauhinia purpurea lectin facilitated the purification of metacyclic promastigotes from stationary-phase culture by negative selection. The B. purpurea non-agglutinated promastigotes had a slender short cell body and long flagella, typical of metacyclic morphology. The ultrastructural analysis showed that B. purpurea non-agglutinated promastigotes have a dense and thicker glycocalyx. They are resistant to complement lysis, and highly infective for macrophage in vitro and hamsters in vivo. Contrary to procyclic promastigotes, B. purpurea non-agglutinated forms were poorly recognised by sand fly gut epithelial cells. These results suggest that the B. purpurea non-agglutinated promastigotes are the metacyclic forms of L. braziliensis.

Introduction

Parasites of the genus Leishmania are digenetic protozoa which exist as amastigotes, forms without free flagella living inside macrophages of the mammalian host, and as promastigotes, which are flagellated forms found in the gut of sand fly vectors. Polymorphic development of promastigotes associated with virulence has been reported in sand flies (Killick-Kendrick, 1979, Sacks and Perkins, 1985) and in axenic cultures (Giannini, 1974, Sacks and Perkins, 1984, Da Silva and Sacks, 1987, Howard et al., 1987, Sacks et al., 1995, Lira et al., 1998). Sequential development from uninfective (procyclic) to infective (metacyclic) stages was first demonstrated to occur in Phlebotomus papatasi infected with Leishmania major (Sacks and Perkins, 1985), where it was observed that only promastigotes obtained 7 days after a blood meal were able to initiate infections in BALB/c mice; before that time they were poorly or non-infective. The same pattern of progressive increase in infectivity has been observed as cultured promastigotes reached the stationary growth phase (Sacks and Perkins, 1984, Howard et al., 1987, Rizvi et al., 1985).

Metacyclogenesis, either in the sand fly gut or in axenic culture medium, is associated not only with morphological changes but also with structural modifications of the promastigote surface lipophosphoglycan. As described for L. major and Leishmania donovani, two Old World species (Da Silva and Sacks, 1987, McConville et al., 1992, Sacks et al., 1995), developmental changes in lipophosphoglycan structure result in down regulation of lectin-binding sites. In both these species, metacyclic promastigotes are purified by negative selection, having lost the ability of being agglutinated by peanut lectin and concanavalin A. Lira et al., 1998, Courret et al., 1999 showed that it is also possible to purify, respectively, Leishmania tropica and Leishmania amazonensis metacyclics from axenic culture using anti-lipophosphoglycan MAb. A lectin or MAb that could similarly be used to purify metacyclics of Leishmania braziliensis has not been found.

Previous work showed that L. braziliensis metacyclic promastigotes were agglutinated by lentil lectin (Almeida et al., 1993), but it is difficult to then use lentil lectin agglutinated forms for further studies. Here, we describe the purification of metacyclic promastigotes of L. braziliensis, the causative agent of mucocutaneous leishmaniasis in the New World by negative selection using Bauhinia purpurea lectin. Our results show that B. purpurea lectin non-agglutinated promastigotes display metacyclic characteristics such as morphology, complement resistance and infectivity in vitro and in vivo.