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Knockdown of LdMC1 and Hsp70 by antisense oligonucleotides causes cell-cycle defects and programmed cell death in Leishmania donovani

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Abstract

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.

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Acknowledgments

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.

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The authors declare no conflict of interest.

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  1. Parasitology Laboratory, Department of Zoology, Panjab University, Chandigarh, 160014, India

    Puneet Raina & Sukhbir Kaur

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  1. Puneet Raina
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Correspondence to Sukhbir Kaur.

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11010_2011_1007_MOESM1_ESM.pdf

ESM 1 Position of designed antisense oligonucleotide on LdMC1 gene. A-On nucleotide sequence, B-on mRNA secondary structure, C-relative location on the mRNA (PDF 503 kb)

11010_2011_1007_MOESM2_ESM.pdf

ESM 2 Position of designed antisense oligonucleotide on Hsp70 gene. A-On Nucleotide sequence, B- On mRNA secondary structure, C-relative location on the mRNA (PDF 543 kb)

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Raina, P., Kaur, S. Knockdown of LdMC1 and Hsp70 by antisense oligonucleotides causes cell-cycle defects and programmed cell death in Leishmania donovani . Mol Cell Biochem 359, 135–149 (2012). https://doi.org/10.1007/s11010-011-1007-y

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