Transgenic, fluorescent Leishmania mexicana allow direct analysis of the proteome of intracellular amastigotes.
Mol Cell Proteomics
; 7(9): 1688-701, 2008 Sep.
Article
en En
| MEDLINE
| ID: mdl-18474515
ABSTRACT
Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to approximately 6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Leishmania mexicana
/
Proteínas Protozoarias
/
Separación Celular
/
Proteómica
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Citometría de Flujo
Tipo de estudio:
Prognostic_studies
Límite:
Animals
País/Región como asunto:
Mexico
Idioma:
En
Revista:
Mol Cell Proteomics
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Año:
2008
Tipo del documento:
Article
País de afiliación:
Reino Unido