Comprehensive Proteomics Analysis of Glycosomes fromLeishmania donovani

Abstract

Leishmania donovani is a kinetoplastid protozoan that causes a severe and fatal disease kala-azar, or visceral leishmaniasis. L. donovani infects human host after the phlebotomine sandfly takes a blood meal and resides within the phagolysosome of infected macrophages. Previous studies on host-parasite interactions have not focused on Leishmania organelles and the role that they play in the survival of this parasite within macrophages. Leishmania possess glycosomes that are unique and specialized subcellular microbody organelles. Glycosomes are known to harbor most peroxisomal enzymes and, in addition, they also possess nine glycolytic enzymes. In the present study, we have carried out proteomic profiling using high resolution mass spectrometry of a sucrose density gradient-enriched glycosomal fraction isolated from L. donovani promastigotes. This study resulted in the identification of 4022 unique peptides, leading to the identification of 1355 unique proteins from a preparation enriched in L. donovani glycosomes. Based on protein annotation, 566 (41.8%) were identified as hypothetical proteins with no known function. A majority of the identified proteins are involved in metabolic processes such as carbohydrate, lipid, and nucleic acid metabolism. Our present proteomic analysis is the most comprehensive study to date to map the proteome of L. donovani glycosomes.

A Global Analysis of Enzyme Compartmentalization to Glycosomes

“…In order to establish a starting population of proteins for which glycosomal localization is well supported by experimental evidence, we performed a meta-analysis that included as many different information types as possible. We included studies in species Trypanosoma cruzi and Leishmania donovani in addition to T. brucei where most work is performed (Figure 1, [10][11][12][19][20][21][22]). These three species, all dixenous mammalian parasites, are still similar in lifestyle relative to the full complement of species containing glycosomes.…”

“…Weighting of these studies was as follows: presence in [19] 1 point if yes, 0 points if no; presence in [22] 0.5 points for each life stage-procyclic and/or bloodstream forms-that it was observed in, 0 points if not found; presence in [20] 2 points if yes, 0 points if no; presence in [12] 2 points if yes, 0 points if no; presence in [11] 2 points if yes, 0 points if no, presence in [10] 2 points if yes, 0 points if no; glycosome localization pattern upon N-terminal tagging in TrypTag [21] 3 points if yes, 0 points if information was not available (tagging on the N-terminus was unsuccessful or not attempted), and −2 points if localization of signal from the N-terminus tagged protein was other than glycosomal (we utilized a lesser negative score for non-glycosomal localization because multiple non-relevant factors can lead to non-targeting of a tagged protein while a false positive is rare). As a C-terminal tagging could obscure a terminal peroxisome targeting signal 1, it was ignored.…”

“…Our understanding of the complement of glycosome-localized metabolic enzymes is centered on experiments cataloguing them in Trypanosoma brucei, and to a lesser extent Trypanosoma cruzi and Leishmania spp.-all dixenous organisms capable of causing human and livestock disease (e.g., [10][11][12]). As the Trypanosoma and Leishmania genera are phylogenetically distinct, the fact of apparent overlap in their glycosome enzyme composition suggests a high degree of conservation [13].…”

Potential Drug Targets in the Pentose Phosphate Pathway of Trypanosomatids

“…Regarding RPI localization, LmRPIB possesses a PTS-2 signal sequence (-RVALGCDHA-), which is conserved in L. infantum and L. donovani [249]. However, recent proteomic analysis of L. donovani glycosomes failed to detect RPIB in these organelles [266]. The same analysis detected HGPRT (PTS-1), aldolase (PTS-2), as well as some enzymes of PPP that are upstream RPIB, namely putative G6PD (PTS-1), putative 6PGDH (non-identified), or enzymes downstream RPIB, such as TKL (PTS-1), putative RPE (PTS-1) and putative TAL (nonidentified signal peptide), or other related proteins like putative ribokinase (PTS-2 signal peptide).…”

Proteomics in India: the clinical aspect

“…These efforts are providing insights into its physiology whereas proteomic analysis of serum from infected individuals gives preliminary information concerning potential biomarkers [26–29]. Several studies have also been conducted on clinical isolates of wild type and drug resistant varieties of Leishmania across different institutes of the country like NCCS, Pune; JNU, New Delhi; CDRI, Lucknow; IOB, Bangalore; AIIMS, New Delhi; NIPER, Mohali; BHU, Varanasi and IICB, Kolkata [30–44]. Interesting findings have also been reported on other infectious diseases like hepatitis, food poisoning, leprosy etc.…”