Proteomic analysis of the secretome of Leishmania donovani.
BACKGROUND
Leishmania and other intracellular pathogens have evolved strategies that support invasion and persistence within host target cells. In some cases the underlying mechanisms involve the export of virulence factors into the host cell cytosol. Previous work from our laboratory identified one such candidate leishmania effector, namely elongation factor-1alpha, to be present in conditioned medium of infectious leishmania as well as within macrophage cytosol after infection. To investigate secretion of potential effectors more broadly, we used quantitative mass spectrometry to analyze the protein content of conditioned medium collected from cultures of stationary-phase promastigotes of Leishmania donovani, an agent of visceral leishmaniasis.


RESULTS
Analysis of leishmania conditioned medium resulted in the identification of 151 proteins apparently secreted by L. donovani. Ratios reflecting the relative amounts of each leishmania protein secreted, as compared to that remaining cell associated, revealed a hierarchy of protein secretion, with some proteins secreted to a greater extent than others. Comparison with an in silico approach defining proteins potentially exported along the classic eukaryotic secretion pathway suggested that few leishmania proteins are targeted for export using a classic eukaryotic amino-terminal secretion signal peptide. Unexpectedly, a large majority of known eukaryotic exosomal proteins was detected in leishmania conditioned medium, suggesting a vesicle-based secretion system.


CONCLUSIONS
This analysis shows that protein secretion by L. donovani is a heterogeneous process that is unlikely to be determined by a classical amino-terminal secretion signal. As an alternative, L. donovani appears to use multiple nonclassical secretion pathways, including the release of exosome-like microvesicles.
Silverman
J Maxwell
JM
Department of Medicine (Division of Infectious Diseases), University of British Columbia, Faculty of Medicine, 2733 Heather St, Vancouver, British Columbia, V5Z 3J5, Canada. maxer@interchnage.ubc.ca
Chan
Simon K
SK
Robinson
Dale P
DP
Dwyer
Dennis M
DM
Nandan
Devki
D
Foster
Leonard J
LJ
Reiner
Neil E
NE
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D019295:Computational Biology; D017077:Culture Media, Conditioned / Q000737:chemistry; D017125:Genes, Protozoan; D007893:Leishmania donovani / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008830:Microbodies / Q000378:metabolism; D040901:Proteomics; D015800:Protozoan Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism
D017077:Culture Media, Conditioned; D015800:Protozoan Proteins