A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.
At the end of their growth in the sand fly, Leishmania parasites differentiate into the infective metacyclic promastigote stage, which is transmitted to the mammalian host. Thus, in experimental studies of parasite infectivity toward animals or macrophages, the use of purified metacyclics is generally preferred. While metacyclics of several Leishmania species can be efficiently purified with the aid of lectins or monoclonal antibodies, which differentially exploit stage-specific differences in the structure of the abundant surface glycolipid lipophosphoglycan (LPG), such reagents are unavailable for most species and they are unsuitable for studies involving LPG-deficient mutants. Here we describe a simple density gradient centrifugation method, which allows the rapid purification of infective metacyclic parasites from both wild-type and LPG-deficient Leishmania major. The purified metacyclic promastigotes are authentic, as judged by criteria such as their morphology, expression of the metacyclic-specific gene SHERP, and ability to invade and replicate within macrophages in vitro. Preliminary studies suggest that this method is applicable to other Leishmania species including L. donovani.
Späth
G F
GF
Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St., Louis, Missouri, 63110, USA.
Beverley
S M
SM
D000372:Agglutination Tests; D000818:Animals; D015152:Blotting, Northern; D002240:Carbohydrate Sequence; D002499:Centrifugation, Density Gradient; D005260:Female; D006028:Glycosphingolipids / Q000737:chemistry*; D018320:Leishmania major / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D008264:Macrophages / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D016053:RNA, Protozoan / Q000032:analysis; D014774:Virulence
D006028:Glycosphingolipids; D016053:RNA, Protozoan; C008290:lipophosphonoglycan