An in vitro system for developmental and genetic studies of Leishmania donovani phosphoglycans.
Glycoconjugates have been shown to play important roles in Leishmania development. However, the ability to study these molecules and other processes would benefit greatly from improved methods for genetic manipulation and analysis of the amastigote stage. This is especially challenging for L. donovani, the agent of the most severe form of leishmaniasis, which can rapidly lose virulence during in vitro culture. Here we report on a clonal subline of an L. donovani 1S2D (LdBob or LdB), which differentiates readily from promastigotes to amastigotes in axenic culture, and maintains this ability during extended parasite cultivation in vitro. This derivative can be plated and transfected efficiently while grown as promastigotes or amastigotes. Importantly, LdB maintains the ability to differentiate while undergoing genetic alterations required for creation of gene knockouts and complemented lines. Like virulent L. donovani, LdB exhibits down-regulation of lipophosphoglycan (LPG) synthesis and up-regulation of A2 protein synthesis in amastigotes. We showed that knockouts of LPG2, encoding a Golgi GDP-mannose transporter, eliminated phosphoglycan synthesis in LdB axenic amastigotes. These and other data suggest that LdB axenic amastigotes will be generally useful as a differentiation model in studies of gene expression, virulence, glycoconjugate function and drug susceptibility in L. donovani.
Goyard
Sophie
S
Department of Molecular Microbiology, Washington University Medical School, Campus Box 8230, 660 S Euclid Ave, St. Louis, MO 63110, USA.
Segawa
Hiroaki
H
Gordon
Jennifer
J
Showalter
Melissa
M
Duncan
Robert
R
Turco
Salvatore J
SJ
Beverley
Stephen M
SM
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D003470:Culture Media; D005786:Gene Expression Regulation; D006028:Glycosphingolipids / Q000235:genetics / Q000378:metabolism*; D007893:Leishmania donovani / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development* / Q000472:pathogenicity; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D021261:Parasitic Sensitivity Tests; D010274:Parasitology / Q000379:methods; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014774:Virulence
D000981:Antiprotozoal Agents; D003470:Culture Media; D006028:Glycosphingolipids; C095909:LPG2 protein, Leishmania; D008565:Membrane Proteins; D015800:Protozoan Proteins; C008290:lipophosphonoglycan