title: Characterization of the proliferating cell nuclear antigen of Leishmania donovani clinical isolates and its association with antimony resistance.
abstract: Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥ 3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ~5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (Sb(V)) and trivalent antimonial (Sb(III)) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of Sb(V) (from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and Sb(III) (from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon Sb(III) treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism.
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Rati
authors.initials: R
authors.lastname: Tandon
authors.affiliation: Division of Structural and Molecular Biology, Central Drug Research Institute, Lucknow, India.
authors.forename: Sharat
authors.initials: S
authors.lastname: Chandra
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Rajendra Kumar
authors.initials: RK
authors.lastname: Baharia
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Sanchita
authors.initials: S
authors.lastname: Das
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Pragya
authors.initials: P
authors.lastname: Misra
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Awanish
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Division of Structural and Molecular Biology, Central Drug Research Institute, Lucknow, India.
authors.forename: Mohammad Imran
authors.initials: MI
authors.lastname: Siddiqi
authors.affiliation: Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
authors.forename: Shyam
authors.initials: S
authors.lastname: Sundar
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India anuradha_dube@hotmail.com.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Dube
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000235:genetics* / Q000378:metabolism; D000967:Antimony Sodium Gluconate / Q000494:pharmacology*; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004351:Drug Resistance; D015870:Gene Expression; D007893:Leishmania donovani / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D007898:Leishmaniasis, Visceral / Q000188:drug therapy* / Q000469:parasitology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics; D017422:Sequence Analysis, DNA
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000981:Antiprotozoal Agents; D018809:Proliferating Cell Nuclear Antigen; D009569:Nitric Oxide; D000967:Antimony Sodium Gluconate