title: A quantitative proteomic screen to identify potential drug resistance mechanism in α-difluoromethylornithine (DFMO) resistant Leishmania donovani.
abstract: Visceral leishmaniasis (VL) caused by Leishmania donovani is a systemic protozoan disease that is fatal if left untreated. The promastigote form of L. donovani is sensitive to growth inhibition by dl-α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania (DFMO-16), which was capable of proliferating in 16mM DFMO. To elucidate the molecular basis for this resistance, we undertook a comparative proteomic analysis of DFMO-resistant/sensitive parasites using isobaric tagging for relative and absolute quantification (iTRAQ/LC-MS/MS). Out of the 101 proteins identified in at least 2 of the 3 independent experiments, 82 proteins are 1.5- to 44.0-fold more abundant in DFMO-resistant strain (DFMO-16) while 19 are 2- to 5.0-fold less abundant as compared to the wild-type (DI700) parasites. Proteins with 2-fold or greater abundance in the DFMO-resistant strain include free radical detoxification, polyamine and trypanothione metabolic proteins, proteins involved in metabolism, intracellular survival and proteolysis, elongation factors, signaling molecules and mitochondrial transporters, and many with no annotated function. Differentially modulated proteins contribute to our understanding of molecular mechanism of DFMO-resistance and have the potential to act as biomarkers.
  
  
  CONCLUSIONS
  This study will facilitate a deeper understanding of the phenomenon of acquired drug resistance and possible biomarkers in Leishmania against antiparasitic drug DFMO. Also it will provide information about the metabolic pathways modulated in resistant parasites as an adaptation mechanism to counter drugs. Studies like this are important to safeguard the efficacy of a limited repertoire of anti-parasitic drugs, and to lead the development of new drugs and drug combinations.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Alok Kumar
authors.initials: AK
authors.lastname: Singh
authors.affiliation: School of Pharmacy, Pacific University, OR, USA.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Roberts
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, USA.
authors.forename: Buddy
authors.initials: B
authors.lastname: Ullman
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India. Electronic address: rentala@outlook.com.
authors.forename: Rentala
authors.initials: R
authors.lastname: Madhubala
mesh_terms: D015415:Biomarkers / Q000737:chemistry; D002853:Chromatography, Liquid; D004351:Drug Resistance; D000518:Eflornithine / Q000737:chemistry*; D005609:Free Radicals; D020558:GTP Phosphohydrolases / Q000737:chemistry; D005978:Glutathione / Q000031:analogs & derivatives / Q000737:chemistry; D007893:Leishmania donovani / Q000378:metabolism*; D013058:Mass Spectrometry; D009955:Ornithine Decarboxylase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry; D017510:Protein Folding; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D013095:Spermidine / Q000031:analogs & derivatives / Q000737:chemistry; D013347:Subcellular Fractions / Q000737:chemistry
chemical_list: D015415:Biomarkers; D005609:Free Radicals; D010455:Peptides; D011073:Polyamines; C044809:trypanothione; D020558:GTP Phosphohydrolases; D009955:Ornithine Decarboxylase; D005978:Glutathione; D013095:Spermidine; D000518:Eflornithine
keywords: Difluoromethylornithine; Drug resistance; Leishmania donovani; Ornithine decarboxylase; Polyamines; Proteomics