Differential quantitative proteomic profiling of Leishmania infantum and Leishmania mexicana density gradient separated membranous fractions.

UNLABELLED: Leishmaniasis, caused by infection with Leishmania, is a major public health concern affecting more than 20million people globally. Leishmania has a digenetic lifecycle consisting of an extracellular flagellated promastigote, adapted to live in the mid-gut of the sand fly host and an aflagellated intracellular amastigote that resides within the macrophage of the mammalian host. Leishmania mexicana and Leishmania infantum are causative agents of cutaneous and visceral leishmaniasis, respectively. Membrane proteins play a pivotal role in host-pathogen interactions and in regulatory pathways. As the genome of Leishmania is essentially constitutively expressed, regulation of protein expression during differentiation occurs post-transcriptionally and/or post-translationally. Quantitative mass spectrometry using iTRAQ labeling identified differences in the proteomes of density gradient separated membranous fractions of promastigote and amastigote life-stages. We identified 189 L. infantum and 107 L. mexicana non-redundant proteins of which 20-40% showed differential expression levels between promastigote and amastigote lifecycle stages. Differentially expressed proteins mapped to several pathways including cell motility, metabolism, and infectivity as well as virulence factors such as eEF-1α, amastin and leishmanolysin (GP63). Western blot analysis validated iTRAQ quantitation for leishmanolysin. Focusing on differentially expressed proteins essential for pathogenesis, may ultimately lead to the identification of novel potential therapeutic targets.

BIOLOGICAL SIGNIFICANCE: Leishmania, protozoan parasites of the Trypanosomatidae family, are the causative agents of leishmaniasis that represents a major public health concern affecting more than 20million people globally Membrane associated proteins play a pivotal role in host-pathogen interactions and in regulatory pathways. Quantitative proteomic analysis of the membranous fractions from L. mexicana and L. infantum (causative agents of cutaneous and visceral leishmaniasis, respectively) identified a number of proteins that may have important stage-specific functions in either the sand fly or mammalian host. The function of these proteins includes roles in virulence, as well as differences in metabolic process between life stages. Many of the proteins identified may act as virulence factors playing significant roles in parasite invasion, host-parasite interaction or parasite survival and thus may have therapeutic potential as drug target candidates.