Knockdown of LdMC1 and Hsp70 by antisense oligonucleotides causes cell-cycle defects and programmed cell death in Leishmania donovani
- 366 Downloads
Programmed cell death (PCD) has important implications in the biology of unicellular parasites, especially in devising control strategies against them. In this study, we examined the role of metacaspase LdMC1 and heat shock protein Hsp70 in Leishmania donovani through transient gene knockdown using antisense oligonucleotides (ASOs), during MG132-induced PCD. Proteasome inhibitor MG132 was used for inducing PCD in the in vitro culture of Leishmania donovani, which was confirmed by morphological and molecular markers. To assess the role of LdMC1 and Hsp70, ASOs with partially modified phosphorothioate backbone were designed against the protein-coding regions of these genes. Promastigotes and axenic ALFs were exposed to ASOs, and gene knockdown was confirmed using RT-PCR. Exposure to MG132 and ASOs led to morphological defects, DNA fragmentation, delay in progressing through the S-phase of cell-cycle and a decrease in the mitochondrial membrane potential. Antisense knockdown of both these genes, individually as well as together, caused phenotypic and molecular characteristics of PCD. Simultaneous knockdown of both LdMC1 and Hsp70 led to a severity in these defects. Parasites co-exposed to MG132 along with ASOs suffered the maximum damage. Together, these data suggest that LdMC1 and Hsp70 have an indispensable role in Leishmania cell-cycle and are, therefore, important for its survival.
KeywordsLeishmania donovani Programmed cell death MG132 Metacaspase Hsp70 Antisense oligonucleotides
PR was supported by Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), India. SK is supported in the form of extramural funding by Indian Council of Medical Research (ICMR), India.
Conflict of interest
The authors declare no conflict of interest.
- 1.World Health Organization (2004) TDR Summary Report 2004–05Google Scholar
- 3.World Health Organization (2002) Weekly epidemiological record. 77:365–370Google Scholar
- 17.Arnoult D, Akarid K, Grodet A, Petit PX, Estaquier J, Amiesen JC (2002) On the evolution of programmed cell death: apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilisation. Cell Death Differ 9:65–81PubMedCrossRefGoogle Scholar
- 45.Alcolea PJ, Alonso A, Gómez MJ, Sánchez-Gorostiaga A, Moreno-Paz M, González-Pastor E, Toraño A, Parro V, Larraga V (2010) Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum. BMC. Genomics 11:31Google Scholar
- 56.Compagno D, Lampe JN, Bourget C, Kutyavin IV, Yurchenko L, Lukhtanov EA, Gorn VV, Gamper HB Jr, Toulmé JJ (1999) Antisense oligonucleotides containing modified bases inhibit in vitro translation of Leishmania amazonensis mRNAs by invading the mini-exon hairpin. J Biol Chem 274:8191–8198PubMedCrossRefGoogle Scholar
- 57.Mishra M, Porter-Kelley JM, Singh PK, Bennett JR, Chaudhuri G (2001) Enhanced activity of antisense phosphorothioate oligos against Leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions. Biochem Pharmacol 62:569–580PubMedCrossRefGoogle Scholar
- 67.Roy A, Ganguly A, BoseDasgupta S, Das BB, Pal C, Jaisankar P, Majumder HK (2008) Mitochondria-dependent reactive oxygen species-mediated programmed cell death induced by 3,3′-diindolylmethane through inhibition of F0F1-ATP synthase in unicellular protozoan parasite Leishmania donovani. Mol Pharmacol 74:1292–1307PubMedCrossRefGoogle Scholar
- 68.Akopyants NS, Matlib RS, Bukanova EN, Smeds MR, Brownstein BH, Stormo GD, Beverley SM (2004) Expression profiling using random genomic DNA microarrays identifies differentially expressed genes associated with three major developmental stages of the protozoan parasite Leishmania major. Mol Biochem Parasitol 136:71–86PubMedCrossRefGoogle Scholar
- 80.Campos RM, Nascimento M, Ferraz JC, Pereira MM, Rocha PO, Thompson GM, Cysne-Finkelstein L, Figueiredo RC, de Melo Neto OP (2008) Distinct mitochondrial HSP70 homologues conserved in various Leishmania species suggest novel biological functions. Mol Biochem Parasitol 160:157–162PubMedCrossRefGoogle Scholar