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.
Highlights
Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material
Leishmania spp. are unicellular parasites of the trypanosomatid family and the causative agents of a spectrum of diseases, the leishmaniases, that affect some 12 million people worldwide [1]. They oscillate between free living, flagellated promastigotes transmitted by blood sucking insect vectors and intracellular, non-flagellated amastigotes
The 32.8-Mbp genome sequence of Leishmania major was completed in 2005 and predicts ϳ8300 ORFs organized in 133 polycistronic units of directional gene clusters spread over 36 chromosomes [4] (GeneDB)
Summary
Growth and Differentiation of L. mexicana—L. mexicana mexicana (MNYC/BZ/62/M379) expressing DsRed [20] were maintained in selective medium as described previously [21]. Processing of Promastigotes and Isolation of Amastigotes from Infected Bone Marrow-derived Macrophages or Lesions—Promastigotes in late logarithmic growth phase were harvested by centrifugation and processed according to Ref. 14. Infected bone marrow-derived macrophages were abraded in homogenization buffer (20 mM HEPES-KOH, pH 7.3, 0.25 M sucrose supplemented with “Complete Mini” (Roche Applied Science)). Sorted parasites were collected in 50-ml tubes containing 5 ml of PBS with protease inhibitors and centrifuged at 1200 ϫ g for 10 min. Supernatant was removed completely, and parasite pellets were lysed in incomplete lysis buffer as described above for promastigotes. The resulting pellet was resuspended in an appropriate volume of PBS containing protease inhibitors and loaded onto the discontinuous sucrose gradient, and parasites were purified as above.
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