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Proteomics assays in leishmania

Yau Wai-Lok, Ulrike Lambertz, Lucie Colineau, Pascale Pescher, Andrea MacDonald, Dorothea Zander, Silke Retzlaff, Julia Eick, Neil E Reiner, Joachim Clos, Gerald F Späth
Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, non-redundant functions of the co-chaperone cyclophilin 40 in L. donovani-infected macrophages. Here we characterized in more detail the virulence defect of cyp40-/- null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40-/- parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome...
May 24, 2016: Journal of Eukaryotic Microbiology
Sanchita Das, Priyanka Shah, Rati Tandon, Narendra Kumar Yadav, Amogh A Sahasrabuddhe, Shyam Sundar, Mohammad Imran Siddiqi, Anuradha Dube
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...
August 2015: PLoS Neglected Tropical Diseases
Mehdi Zarean, Sharif Maraghi, Homa Hajjaran, Mehdi Mohebali, Mohammad Hossein Feiz-Hadad, Mohammad Ali Assarehzadegan
BACKGROUND: In this study, two-dimensional gel electrophoresis (2-DE) method was applied to determine and compare the protein spots expressed in the two field isolates of Leishmania major and recovered from the patients who were clinically sensitive and resistant to Glucantime® treatment. METHODS: Leishmania parasites were isolated from the cutaneous lesions of two CL infected patients in Shiraz, south of Iran. The species of the two isolates were identified as L...
January 2015: Iranian Journal of Parasitology
Dan Zilberstein
iTRAQ is a high coverage quantitative proteomics technique identifies and quantitates abundance changes of multiple (up to eight) distinct protein samples. To date, one iTRAQ-MS/MS assay can identify up to quarter of cells proteome. Each of the eight tags covalently binds to the N-terminus as well as arginine and lysine side chains of peptides, enabling labeling of the entire peptide population in each sample. Following tagging, the various protein samples are mixed and subjected to LC-MS/MS analysis. In the first round identical peptides from the different protein populations focus in a single pick...
2015: Methods in Molecular Biology
Micheline Soares Braga, Leandro Xavier Neves, Jonatan Marques Campos, Bruno Mendes Roatt, Rodrigo Dian de Oliveira Aguiar Soares, Samuel Leôncio Braga, Daniela de Melo Resende, Alexandre Barbosa Reis, William Castro-Borges
The exoproteome of some Leishmania species has revealed important insights into host-parasite interaction, paving the way for the proposal of novel disease-oriented interventions. The focus of the present investigation constituted the molecular profile of the L. infantum exoproteome revealed by a shotgun proteomic approach. Promastigotes under logarithmic phase of growth were obtained and harvested by centrifugation at different time points. Cell integrity was evaluated through the counting of viable parasites using propidium iodide labeling, followed by flow cytometry analysis...
June 2014: Molecular and Biochemical Parasitology
Jessica L Martin, Phillip A Yates, Radika Soysa, Joshua F Alfaro, Feng Yang, Kristin E Burnum-Johnson, Vladislav A Petyuk, Karl K Weitz, David G Camp, Richard D Smith, Phillip A Wilmarth, Larry L David, Gowthaman Ramasamy, Peter J Myler, Nicola S Carter
The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity...
February 2014: PLoS Pathogens
Brianna Norris-Mullins, Kaitlin VanderKolk, Paola Vacchina, Michelle V Joyce, Miguel A Morales
We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in a phosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidase member of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. A null mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene in Leishmania major. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigote stages...
2014: PLoS Neglected Tropical Diseases
Eduardo Sobarzo-Sánchez, Pablo Bilbao-Ramos, Maria Dea-Ayuela, Humberto González-Díaz, Matilde Yañez, Eugenio Uriarte, Lourdes Santana, Victoria Martínez-Sernández, Francisco Bolás-Fernández, Florencio M Ubeira
Leishmaniasis is a growing health problem worldwide. As there are certain drawbacks with the drugs currently used to treat human leishmaniasis and resistance to these drugs is emerging, there is a need to develop novel antileishmanial compounds, among which isoquinoline alkaloids are promising candidates. In this study, 18 novel oxoisoaporphine derivatives were synthesized and their possible antileishmanial activity was evaluated. The in vitro activity of these derivatives against Leishmania amazonensis axenic amastigotes was first evaluated, and the selected compounds were then tested in an inhibition assay with promastigotes of L...
2013: PloS One
Fabiana S V Matrangolo, Daniel B Liarte, Laila C Andrade, Melina F de Melo, Juvana M Andrade, Rafael F Ferreira, André S Santiago, Carlos P Pirovani, Rosiane A Silva-Pereira, Silvane M F Murta
The emergence of drug-resistant Leishmania species is a significant problem in several countries. A comparative proteomic analysis of antimony-susceptible and antimony-resistant Leishmania braziliensis (LbSbR) and Leishmania infantum chagasi (LcSbR) lines was carried out using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (LC/MS/MS) for protein identification. Out of 132 protein spots exclusive or up-regulated submitted to MS, we identified 80 proteins that corresponded to 57 distinct proteins...
August 2013: Molecular and Biochemical Parasitology
Adam Sateriale, Kovi Bessoff, Indra Neil Sarkar, Christopher D Huston
OBJECTIVE: To identify potential opportunities for drug repurposing by developing an automated approach to pre-screen the predicted proteomes of any organism against databases of known drug targets using only freely available resources. MATERIALS AND METHODS: We employed a combination of Ruby scripts that leverage data from the DrugBank and ChEMBL databases, MySQL, and BLAST to predict potential drugs and their targets from 13 published genomes. Results from a previous cell-based screen to identify inhibitors of Cryptosporidium parvum growth were used to validate our in-silico prediction method...
March 2014: Journal of the American Medical Informatics Association: JAMIA
Alexandros Alexandratos, Joachim Clos, Martina Samiotaki, Antonia Efstathiou, George Panayotou, Ketty Soteriadou, Despina Smirlis
Overexpression of Leishmania histone H1 (LeishH1) was previously found to cause a promastigote-to-amastigote differentiation handicap, deregulation of cell-cycle progression, and loss of parasite infectivity. The aim of this study was to identify changes in the proteome of LeishH1 overexpressing parasites associated with the avirulent phenotype observed. 2D-gel electrophoresis analysis revealed only a small protein subset of differentially expressed proteins in the LeishH1 overexpressing promastigotes. Among these was the chaperone HSP83, known for its protective role in Leishmania drug-induced apoptosis, which displayed lower translational rates...
June 2013: Molecular Microbiology
Inés Martín-Martín, Ricardo Molina, Maribel Jiménez
In the Indian subcontinent visceral leishmaniasis, also known as kala-azar, is caused by the protozoa Leishmania donovani and is transmitted to humans by the bite of infected female sand flies Phlebotomus argentipes in an anthroponotic cycle. Sand fly saliva is known to play an important role in host infection outcome after an infective bite. Immunogenicity of P. argentipes saliva has already been described. However, specific antigens that can contribute to these immunogenic properties are unknown. This work focuses on the identification of antigens present in P...
June 2013: Acta Tropica
Pramod Kumar Kushawaha, Reema Gupta, Chandra Dev Pati Tripathi, Shyam Sundar, Anuradha Dube
In Leishmania species, Protein disulfide isomerase (PDI)--a redox chaperone, is reported to be involved in its virulence and survival. This protein has also been identified, through proteomics, as a Th1 stimulatory protein in the soluble lysate of a clinical isolate of Leishmania donovani (LdPDI). In the present study, the molecular characterization of LdPDI was carried out and the immunogenicity of recombinant LdPDI (rLdPDI) was assessed by lymphocyte proliferation assay (LTT), nitric oxide (NO) production, estimation of Th1 cytokines (IFN-γ and IL-12) as well as IL-10 in PBMCs of cured/endemic/infected Leishmania patients and cured L...
2012: PloS One
Dhiraj Kumar, Ruchi Singh, Vasundhra Bhandari, Arpita Kulshrestha, Narendra Singh Negi, Poonam Salotra
Resistance to antimony is a major cause of failure to therapy in a large proportion of visceral leishmaniasis cases. Methods to distinguish resistant and sensitive parasite are urgently needed as the standard in vitro intracellular drug susceptibility assays are cumbersome and time consuming. Differential expression profiling studies have led to the identification of several antimony resistance-associated genes; however, their efficacy as a potential biomarker for monitoring antimony resistance remains imprecise...
July 2012: Parasitology Research
Hamid Daneshvar, Susan Wyllie, Stephen Phillips, Paul Hagan, Richard Burchmore
We have previously described an attenuated line of Leishmania infantum (H-line), selected by culturing promastigotes in vitro in the presence of gentamicin. To elucidate the molecular basis for this attenuation, we undertook a comparative proteomic analysis using multiplex 2-dimensional (2D) difference gel electrophoresis. Eighteen proteins that showed significant and reproducible changes in expression were identified. Many of these were components of the thiol-redox control system in Leishmania and this observation, validated by Western blot, prompted us to investigate the sensitivity of the attenuated line to oxidative stress...
February 16, 2012: Journal of Proteomics
Bhavna Chawla, Anupam Jhingran, Aswini Panigrahi, Kenneth D Stuart, Rentala Madhubala
Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL) and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr) strain of L...
2011: PloS One
Oleksiy Kovtun, Sergey Mureev, WooRam Jung, Marta H Kubala, Wayne Johnston, Kirill Alexandrov
Cell-free protein expression is an important tool for a rapid production, engineering and labeling of recombinant proteins. However the complex protocols for preparation of eukaryotic cell-free protein expression systems result in high manufacturing costs and limit their utility. Recently we reported a novel cell-free expression system based on the lysate of a fermentable protozoan Leishmania tarentolae. Herein we describe a protocol for high throughput protein expression using Leishmania cell-free lysate. The protocol combines PCR-based synthesis and engineering of translation templates with a combined transcription-translation system...
September 2011: Methods: a Companion to Methods in Enzymology
Dirk Schmidt-Arras, Olivier Leclercq, Pier Federico Gherardini, Manuela Helmer-Citterich, Wolfgang Faigle, Damarys Loew, Gerald F Späth
The protozoan parasite Leishmania donovani undergoes various developmental transitions during its infectious cycle that are triggered by environmental signals encountered inside insect and vertebrate hosts. Intracellular differentiation of the pathogenic amastigote stage is induced by pH and temperature shifts that affect protein kinase activities and downstream protein phosphorylation. Identification of parasite proteins with phosphotransferase activity during intracellular infection may reveal new targets for pharmacological intervention...
August 24, 2011: Journal of Proteomics
Subodh Kumar, Dinesh Kumar, Jaya Chakravarty, Shyam Sundar
The biggest challenge in the serological diagnosis of visceral leishmaniasis (VL) is to find a biomarker with a high specificity. This study was undertaken to identify novel Leishmania donovani antigens to solve the existing problem. The soluble L. donovani promastigote antigen was separated by SDS-PAGE, and a Western blot was probed with pooled sera of five subjects with confirmed VL before (n = 9 pools) and after (n = 9 pools) treatment and at the 6-month follow-up visit (n = 9 pools), healthy controls not from an area of endemicity (n = 9 pools), and healthy controls from an area of endemicity...
May 2011: Clinical and Vaccine Immunology: CVI
Yasuyuki Goto, Darrick Carter, Jeffrey Guderian, Noboru Inoue, Shin-Ichiro Kawazu, Steven G Reed
Proteins with tandem repeat (TR) domains have been found in various protozoan parasites, and they are often targets of B-cell responses. Through systematic analyses of whole proteomes, we recently demonstrated that two trypanosomatid parasites, Leishmania infantum and Trypanosoma cruzi, are rich in antigenic proteins with large TR domains. However, the reason that these proteins are antigenic was unclear. Here, by performing molecular, immunological, and bioinformatic characterizations of Leishmania TR proteins, we found two possible factors affecting the antigenicity of these proteins; one factor is their fundamental composition as TR proteins, and the other is regulation of their expression by parasites...
May 2010: Infection and Immunity
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