Expression of the major surface glycoprotein of Leishmania, gp63, in wild-type and sinefungin-resistant promastigotes.
In this study, we have surveyed gp63 expression in sinefungin-(SF)-resistant and wild-type Leishmania promastigotes. Documentation of gp63 expression in Leishmania promastigotes was carried out by Western blotting, purification of the protein and assessment of gp63 protease activity. We demonstrated a 3-4-fold and 1.5-2-fold increase of gp63 protein in SF-resistant Leishmania donovani and Leishmania tropica promastigotes compared to wild-type, respectively. Northern blot analysis showed that the increase in the amount of gp63 protein in SF-resistant compared to wild-type parasites was concomitant with an increase in gp63 mRNA. No extrachromosomal DNA was identified by alkaline lysis of isolated DNA samples and Southern blot analysis. Treatment of SF-resistant and wild-type L. donovani promastigotes with cycloheximide resulted in an increase of the steady state levels of gp63 mRNA in the SF-resistant parasites to approximately fivefold that of the wild type. After treating parasites with actinomycin D, estimated gp63 mRNA t1/2 in the wild type was 40 min and increased to 83 min in SF-resistant promastigotes. Therefore, the overexpression of gp63 may be mediated, at least in part, by post-transcriptional stabilization of a gp63 transcript by a protein factor. Down regulation of the latter factor may account for the observed increase in gp63 expression in SF-resistant promastigotes. Attempts to correlate gp63 expression with promastigote virulence suggested that the observed increase in gp63 expression did not result in a significant change in the virulence of SF-resistant compared to wild-type L. donovani promastigotes.
Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece.
K P
KP
Soteriadou
A K
AK
Tzinia
A
A
Mamalaki
M A
MA
Phelouzat
F
F
Lawrence
M
M
Robert-Gero
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D004351:Drug Resistance; D010450:Endopeptidases / Q000378:metabolism; D007891:Leishmania / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D007896:Leishmaniasis / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism
D000981:Antiprotozoal Agents; D015800:Protozoan Proteins; D012333:RNA, Messenger; D010450:Endopeptidases; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania; D000241:Adenosine; C006235:sinefungin