Abstract
Adaptation to starvation is integral to the Leishmania life cycle. The parasite can survive prolonged periods of nutrient deprivation both in vitro and in vivo. The identification of parasite proteins synthesised during starvation is key to unravelling the underlying molecular mechanisms facilitating adaptation to these conditions. Additionally, as stress adaptation mechanisms in Leishmania are linked to virulence as well as infectivity, profiling of the complete repertoire of Newly Synthesised Proteins (NSPs) under starvation is important for drug target discovery. However, differential identification and quantitation of low abundance, starvation-specific NSPs from the larger background of the pre-existing parasite proteome has proven difficult, as this demands a highly selective and sensitive methodology. Herein we introduce an integrated chemical proteomics method in L. mexicana promastigotes that involves a powerful combination of the BONCAT technique and iTRAQ quantitative proteomics Mass Spectrometry (MS), which enabled temporally resolved quantitative profiling of de novo protein synthesis in the starving parasite. Uniquely, this approach integrates the high specificity of the BONCAT technique for the NSPs, with the high sensitivity and multiplexed quantitation capability of the iTRAQ proteomics MS. Proof-of-concept experiments identified over 250 starvation-responsive NSPs in the parasite. Our results show a starvation-specific increased relative abundance of several translation regulating and stress-responsive proteins in the parasite. GO analysis of the identified NSPs for Biological Process revealed translation (enrichment P value 2.47e-35) and peptide biosynthetic process (enrichment P value 4.84e-35) as extremely significantly enriched terms indicating the high specificity of the NSP towards regulation of protein synthesis. We believe that this approach will find widespread use in the study of the developmental stages of Leishmania species and in the broader field of protozoan biology.
Highlights
Protozoan parasites of the Leishmania spp. are the causative agents of leishmaniasis, a Neglected Tropical Disease (NTD) endemic in over 90 countries worldwide, affecting approximately 12 million people with an estimated 700,000 to 1 million new cases annually [1]
Differential identification of the complete repertoire of these proteins synthesised during starvation from the pre-existing proteins in the parasite holds the key to understanding the starvation adaptation mechanisms
Using a combination of chemical labelling techniques and protein mass-spectrometry, we selectively identified and measured the proteins generated in the starving Leishmania parasite
Summary
Protozoan parasites of the Leishmania spp. are the causative agents of leishmaniasis, a Neglected Tropical Disease (NTD) endemic in over 90 countries worldwide, affecting approximately 12 million people with an estimated 700,000 to 1 million new cases annually [1]. These protozoa have a complex life cycle, progressing from extracellular promastigote stages in the sandfly vector to the obligate intramacrophage amastigote stage in the mammalian host [2]. The key proteins involved in the starvation-adaptation mechanisms of the parasite remains unknown and the identification and quantitation of proteins synthesised de novo during starvation is critical to develop understanding of these. We describe a combination of the bio-orthogonal non-canonical amino acid tagging (BONCAT) [6,7] technology and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass-spectrometry (MS) proteomics [8,9] to quantitatively profile the newly synthesised proteome (NSP) of Leishmania mexicana promastigotes during starvation
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