title: Over-Expression of Cysteine Leucine Rich Protein Is Related to SAG Resistance in Clinical Isolates of Leishmania donovani.
abstract: BACKGROUND
  Resistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance.
  
  
  RESULTS
  In pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed.
  
  
  CONCLUSIONS
  The study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche. Results indicates the possible contribution of CLrP to antimonial resistance in L. donovani by assisting the parasite growth in the macrophages.
authors: Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Sanchita
authors: S
authors: Das
authors: Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Priyanka
authors: P
authors: Shah
authors: Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Rati
authors: R
authors: Tandon
authors: Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Narendra Kumar
authors: NK
authors: Yadav
authors: Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Amogh A
authors: AA
authors: Sahasrabuddhe
authors: Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
authors: Shyam
authors: S
authors: Sundar
authors: Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Mohammad Imran
authors: MI
authors: Siddiqi
authors: Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Anuradha
authors: A
authors: Dube
mesh_terms: D000913:Antibodies, Protozoan; D000965:Antimony / Q000494:pharmacology*; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance; D004926:Escherichia coli; D005786:Gene Expression Regulation; D018503:Genome, Protozoan; D007893:Leishmania donovani / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000913:Antibodies, Protozoan; D000981:Antiprotozoal Agents; D016054:DNA, Protozoan; D015800:Protozoan Proteins; C034426:sodium antimonate; D000965:Antimony