Leishmania EF-1alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation.
The human leishmaniasis are persistent infections of macrophages caused by protozoa of the genus Leishmania. The chronic nature of these infections is in part related to induction of macrophage deactivation, linked to activation of the Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of SHP-1 activation, lysates of Leishmania donovani promastigotes were subjected to SHP-1 affinity chromatography and proteins bound to the matrix were sequenced by mass spectrometry. This resulted in the identification of Leishmania elongation factor-1alpha (EF-1alpha) as a SHP-1-binding protein. Purified Leishmania EF-1alpha, but not host cell EF-1alpha, bound directly to SHP-1 in vitro leading to its activation. Three independent lines of evidence indicated that Leishmania EF-1alpha may be exported from the phagosome thereby enabling targeting of host SHP-1. First, cytosolic fractions prepared from macrophages infected with [(35)S]methionine-labeled organisms contained Leishmania EF-1alpha. Second, confocal, fluorescence microscopy using Leishmania-specific antisera detected Leishmania EF-1alpha in the cytosol of infected cells. Third, co-immunoprecipitation showed that Leishmania EF-1alpha was associated with SHP-1 in vivo in infected cells. Finally, introduction of purified Leishmania EF-1alpha, but not the corresponding host protein into macrophages activated SHP-1 and blocked the induction of inducible nitric-oxide synthase expression in response to interferon-gamma. Thus, Leishmania EF-1alpha is identified as a novel SHP-1-binding and activating protein that recapitulates the deactivated phenotype of infected macrophages.
Nandan
Devki
D
Department of Medicine, Division of Infectious Diseases, The University of British Columbia, Research Institute of the Vancouver Hospital and Health Sciences Center, Vancouver, British Columbia V5Z 3J5, Canada. dnandan@interchange.ubc.ca
Yi
Taolin
T
Lopez
Martin
M
Lai
Crystal
C
Reiner
Neil E
NE
D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D007893:Leishmania donovani / Q000378:metabolism* / Q000472:pathogenicity; D008264:Macrophages / Q000378:metabolism* / Q000469:parasitology; D013058:Mass Spectrometry; D051379:Mice; D008856:Microscopy, Fluorescence; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism*; D018909:src Homology Domains
D047908:Intracellular Signaling Peptides and Proteins; D020648:Peptide Elongation Factor 1; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D005982:Glutathione Transferase; D054645:Protein Phosphatase 1; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C509248:Ptpn6 protein, mouse