RESUMO
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.
Assuntos
Leishmania infantum/metabolismo , Leishmania mexicana/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Proteínas de Protozoários/metabolismo , Humanos , Leishmaniose Cutânea/metabolismo , Leishmaniose Visceral/metabolismo , Especificidade da EspécieRESUMO
Leishmania parasites, the causative agent of leishmaniasis, have a digenetic lifecycle consisting of the morphologically distinct insect vector stage (promastigote) and the mammalian infective amastigote stage. Differentiation of promastigotes to the amastigote stage involves significant morphological and biochemical changes, however, very few genes have been characterised as being differentially expressed in the two stages. The Leishmania A600 genes are one of the few gene families that exhibit stage-specific expression and, as such, they are of interest as potential virulent factors. In this study, we characterize the A600 family in several Leishmania species and investigate their role in amastigote differentiation and proliferation. Four open reading frames, A600-1, A600-2, A600-3, and A600-4, were identified at the multi-gene L. mexicana A600 locus via cloning and restriction mapping. Homology searching identified A600 homologues in other Leishmania species, L. major, L. braziliensis and L. infantum but not in the closely related Trypanosoma family. A targeted gene deletion approach was utilized to determine the cellular function of the L. mexicanaA600 genes. A600(-/-) promastigotes differentiated to axenic amastigotes in response to temperature shift and acidification of culture media, but showed significant growth arrest. Similarly, during in vitro macrophage infection studies, A600(-/-) promastigotes established an early infection, but were deficient in their ability to proliferate as intracellular amastigotes. The ability of A600(-/-) amastigotes to proliferate in mouse peritoneal macrophages was restored by re-introduction of the A600-1 gene, but not the A600-4 gene. The results from these experiments show that the A600-1 gene is essential for continued proliferation of amastigotes, and potentially for development of chronic leishmaniasis. Furthermore, these results suggest a potential role for the L. mexicana A600-deficient mutant as a vaccine candidate.