title: Integrating genomics and proteomics permits identification of immunodominant antigens associated with drug resistance in human visceral leishmaniasis in India.
abstract: Resistance of human pathogens like Leishmania to drugs is a growing concern where the multidrug-resistant phenotype renders chemotherapy ineffective. The acquired resistance of Leishmania to antimony has promoted intense research on the mechanisms involved but the question has not been resolved yet. In this study we have explored host-pathogen- drug interactions leading to identification of pharmacological determinants of host macrophages that resist the sodium antimony gluconate (SAG) mediated intracellular parasite killing. mRNA profiling of mammalian host stage amastigotes of sodium antimony gluconate (SAG) 'sensitive' and 'resistant' parasite lines was carried out using Affymetrix GeneChip®Human Genome U133 Plus 2.0 Array. Patient sera was used to identify immunogenic proteins by two-dimensional gel analysis (2DE) and mass spectrometric analysis (LC-MS/MS). Immunofluorescence microscopy confirmed the identities on 'sensitive' and 'resistant' parasite lines. A total of nine immunogenic proteins whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins are altered in expression levels in the 'resistant' parasites. Global expression profiling using microarrays revealed this regulation was not reflected by changes in the levels of the cognate mRNAs. Following identification of proteins by mass spectrometry, one such regulated protein, enolase, was chosen for more detailed analysis. Immunofluorescence microscopy employing antisera against this enzyme confirmed that its level was differentially regulated in the 'resistant' isolate. We show that high serum level of immunoreactive protein is associated with 'resistant' phenotype. Differentially expressed proteins with immunomodulatory activities were found to be associated with the 'resistant phenotype'.
authors.affiliation: Biochemistry Division, CSIR-Central Drug Research Institute, B 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India. Electronic address: neeloo888@yahoo.com.
authors.forename: Neeloo
authors.initials: N
authors.lastname: Singh
authors.affiliation: Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221 005, India. Electronic address: drshyamsundar@hotmail.com.
authors.forename: Shyam
authors.initials: S
authors.lastname: Sundar
mesh_terms: D000953:Antigens, Protozoan / Q000032:analysis*; D000967:Antimony Sodium Gluconate / Q000494:pharmacology; D000981:Antiprotozoal Agents / Q000494:pharmacology; D015153:Blotting, Western; D004351:Drug Resistance / Q000276:immunology*; D015180:Electrophoresis, Gel, Two-Dimensional; D023281:Genomics; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D016056:Immunodominant Epitopes / Q000032:analysis*; D007074:Immunoglobulin G / Q000276:immunology; D007194:India; D007893:Leishmania donovani / Q000187:drug effects* / Q000276:immunology; D007898:Leishmaniasis, Visceral / Q000188:drug therapy / Q000276:immunology / Q000469:parasitology*; D008264:Macrophages / Q000276:immunology / Q000469:parasitology; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D040901:Proteomics; D015800:Protozoan Proteins / Q000276:immunology
chemical_list: D000953:Antigens, Protozoan; D000981:Antiprotozoal Agents; D007106:Immune Sera; D016056:Immunodominant Epitopes; D007074:Immunoglobulin G; D015800:Protozoan Proteins; D000967:Antimony Sodium Gluconate
keywords: Antibodies; Clinical isolates; Drug resistance; Leishmania donovani