CIHR-UBC Strategic Training Program for
Translational Research in Infectious Diseases
Address: D452 – 2733 Heather Pavilion, Vancouver, BC V5Z 3J5
E-mail: tonychow@interchange.ubc.ca
Voice: (604) 875-5063 Fax: (604) 875-4013 Website: http://cmdr.ubc.ca/trainingprogram
Research Theme:
Molecular pathogenesis, innate immunity, cell signalling, immunomodulation
Sub-theme:
Disruption of innate immunity during intracellular infection
Principal Investigator:
Dr. Neil E. Reiner, MD
Telephone:
Fax:
E-mail:
(604) 875-4588
(604) 875-4013
ethan@interchange.ubc.ca
The broad objective of this proposal is to identify novel molecular mechanisms leading to macrophage deactivation
during chronic intracellular infection. Leishmania donovani causes visceral leishmaniasis and is a major cause of
morbidity and mortality. Macrophages infected with L. donovani show defective intracellular signalling and a
phenotype consistent with a deactivated state. This state is associated with increased activity of the Src-homology 2
domain containing phosphotyrosine phosphatase-1 (SHP-1) and the deactivated phenotype is reversed by
phosphotyrosine phosphatase inhibition. In addition, SHP-1 deficient mice and macrophages show phenotypes of
resistance to leishmania infection. Together, these findings establish a role for SHP-1 in leishmania pathogenesis. In
experiments investigating the mechanism of SHP-1 activation by leishmania, elongation factor-1alpha (EF-1a) of L.
donovani was identified as a specific SHP-1 binding and activating protein. Moreover, introduction of leishmania EF-1a
into cells recapitulated the deactivated phenotype of leishmania infected macrophages. This suggests the hypothesis that
negative signalling, cell deactivation and persistent infection in leishmania infected macrophages is related to activation
of SHP-1 by EF-1a. Four specific aims will test this hypothesis. In Aim 1, the EF-1a cDNA from L. donovani will be
cloned and expressed in baculovirus to examine SHP-1 activation by recombinant EF-1a, and to identify functional
domains by site-directed mutagenesis. Aim 2 involves genomic cloning of L. donovani EF-1a, examination of
chromosomal organization, construction of cassettes containing mutated EF-1a for gene replacement, allelic exchange in
L. donovani and characterization of mutant phenotypes in BALB/c mice. Aim 3 will examine the export of EF-1 and
other proteins from the leishmania phagosome using cells infected with 35S-methionine-labeled leishmania followed by
cell fractionation, 2-D SDS-PAGE and protein microsequencing. Specific antisera combined with laser scanning
confocal microscopy will be used to immunolocalize exported proteins in infected cells. In Aim 4, biochemical and
genetic approaches including the modified, yeast two-hybrid system will be used to identify other candidate SHP-1
activator(s) from L. donovani. This research will provide important new knowledge about molecular mechanisms of
intracellular pathogenesis.
Updated: August 2003
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