Cell-based assay to measure Apicoplast Disruption using Imaging
Assay Overview: The parasite apicoplast is an organelle of cyanobacterial origin that employs a prokaryotic transcription and translation machinery. During asexual blood stage development, this organelle adopts an elongated and branched structure as the parasite matures into its trophozoite form, then segregates into individual organelles that parse with the individual daughter merozoites prior more ..
Keywords: malaria, apicoplast, gene expression, Plasmodium falciparum
Assay Overview: The parasite apicoplast is an organelle of cyanobacterial origin that employs a prokaryotic transcription and translation machinery. During asexual blood stage development, this organelle adopts an elongated and branched structure as the parasite matures into its trophozoite form, then segregates into individual organelles that parse with the individual daughter merozoites prior to their release from the infected erythrocyte. To verify if the lead compounds interfere with the apicoplast, we used a whole cell imaging screen with a recombinant parasite line that has GFP-labeled apicoplasts. Parasites were treated with the test compounds at 3 x their IC50 value for four days. The infected cells were then stained with a Hoechst DNA dye to allow the parasite developmental stage to be ascertained. The parasites were also stained with mito-tracker red for comparative purposes. Parasites treated with compounds that disrupt the apicoplast demonstrate diffuse GFP staining, but intact mitochondrial staining. Parasites treated with fast acting compounds generally display poor mito-tracker staining, but intact apicoplast structures.
Expected Outcome: Antibiotics known to interfere with the apicoplast, such as azithromycin and doxycycline, have been observed to disrupt the morphology of the apicoplast in this GFP expressing line. While GFP is still produced in parasites treated with these drugs it is distributed throughout the cytoplasm. This difference in GFP distribution can be measured by comparing the Standard Deviation of the GFP signal between treated and untreated parasites. Parasites with intact apicoplasts have condensed regions of bright GFP staining with almost no signal detectable in the cytoplasm, leading to a high standard deviation. Parasites with disrupted apicoplasts have uniform levels of GFP staining throughout the cytoplasm leading to low Standard Deviations.
Parasites expressing GFP targeted to the apicoplast, 3D7 ACP(L)-GFP, were treated with the indicated compounds in 200 uL of culture media at a 1% hematocrit. Azithromycin was used as a positive control, while parasites treated with drug vehicle (medium containing 0.05 - 0.1% DMSO) served as a negative control. Assays were started with synchronized ring stages, and parasites were harvested on day four. At 48 hrs, 30ul of culture was harvested and stained with Hoechst dye (1ug/ml) and Mitotracker-red (20nM) (Invitrogen) for 30 min at 37 degrees C. Stained parasites were washed, deposited on poly-lysine coated slides and imaged on a Nikon Ti inverted microscope with a 100x objective.
Expected outcome: An active compound will lead to a decreasing numerical value.
Normalization was applied to the raw data signals such that an activity score of 100 is most active and a value of 0 is inactive. Data was compared to the positive control, azithromycin.
PATTERN CORRECTION: No plate pattern correction algorithm was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
For this imaging assay, compounds were not tested at all doses. Instead, apicoplast morphology was assessed at a concentration 2 to 10 fold higher than the IC50 values from previous screening assays. Data was determined with imaging analysis software to determine raw values.
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
b) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Categorized Comment - additional comments and annotations
Data Table (Concise)