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:
Pathogenomics, proteomics, vaccine development, diagnostics
Sub-theme:
Microarray gene analysis induced by Leishmania infection
Principal Investigator:
Dr. W.R. McMaster, DPhil
Telephone:
Fax:
E-mail:
604-875-4134
604-875-5606
robm@interchange.ubc.ca
Leishmania is an intracellular protozoan parasite of humans that infects cells of the mononuclear phagocyte lineage
causing a spectrum of diseases collectively referred to a leishmaniasis. A genomic approach using DNA microarray
technology will be used to analyze gene expression in infected host cells will provide invaluable information of both
host response mechanisms to intracellular pathogens and pathogen induced mechanism to increase survival. The full
range of macrophage genes modulated by leishmania has never been studied comprehensively. Examples of novel genes
that might be discovered using the microarray technology include those encoding novel cytokines, transcription factors,
inhibitors of macrophage activation or molecules involved in vacuole formation within the infected macrophage The
studies proposed also offer the potential to identify the most complete range of macrophage genes induced in response to
Leishmania to date. Specific Aims are:
1.
2.
To identify and analyze macrophage genes that are either induced or repressed by infection with virulent
Leishmania donovani in the presence or absence of IFN-.
To identify macrophage genes, modulated by infection, that may contribute to leishmania pathogenesis by
identifying the difference in expression of genes modulated by infection with either L. major or L. donovani, two
species that result in distinct disease symptoms and host responses.
Human DNA microarrays will be used to identify macrophage genes that are either induced or repressed by infection
with virulent L. donovani as these modulated genes are likely involved with parasite induced suppression of host
response or host defense. Human peripheral blood derived macrophages will be infected with L. donovani lesion
amastigotes, at various times will be RNA isolated (24, 48, 72 hr post infection) and used to synthesize fluorescent
probes for hybridization to the microarrays. Induced or repressed cDNAs will be identified by differential fluorescence.
To identify genes that respond to live Leishmania, controls will consist of heat killed Leishmania and latex bead for
phagocytosis alone. This approach will identify macrophage genes that may be involved with activation, host response
or pathogen survival and may include transcription factors, inhibitors of macrophage activation, or molecules involved
in vacuole formation within the infected macrophage. To identify IFN- responsive genes that are modulated by
infection, macrophages will be infected with L. donovani and 24 hr stimulated with IFN- and then RNA isolated 24, 48,
72 hr later. Comparison of macrophage gene expression in cells stimulated IFN- or controls will identify IFN-
responsive genes that are modulated by intracellular infection.
To focus on the identification of macrophages genes involved in pathogenesis, DNA microarray technology will be used
to identify the differential gene expression in response to two distinct species of Leishmania. This will be done by
comparing the pattern of host cell gene expression, as described above, by infection with virulent L. major, that causes
cutaneous leishmaniasis to the effects observed with infection with L. donovani, that causes visceral leishmaniasis.
These comparisons will provide insight into macrophage gene expression that are uniquely modified by distinct species
of Leishmania and will identify contributing factors to disease pathology or leishmania pathogenesis.
Significance
Microarray analysis of gene expression in infected macrophages is a powerful tool to unravel novel pathways of host
responses common to many intracellular pathogens.
Updated: August 2003
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