Resistance to the cytostatic activity of the antimalarial drug chloroquine (CQ) is becoming well understood, however, resistance to cytocidal effects of CQ is largely unexplored. We find that PfCRT mutations that almost fully recapitulate P. falciparum cytostatic CQ resistance (CQR(CS)) as quantified by CQ IC50 shift, account for only 10-20% of cytocidal CQR (CQR(CC)) as quantified by CQ LD50 shift. Quantitative trait loci (QTL) analysis of the progeny of a chloroquine sensitive (CQS; strain HB3)×chloroquine resistant (CQR; strain Dd2) genetic cross identifies distinct genetic architectures for CQR(CS) vs CQR(CC) phenotypes, including identification of novel interacting chromosomal loci that influence CQ LD50. Candidate genes in these loci are consistent with a role for autophagy in CQR(CC), leading us to directly examine the autophagy pathway in intraerythrocytic CQR parasites. Indirect immunofluorescence of RBC infected with synchronized CQS vs CQR trophozoite stage parasites reveals differences in the distribution of the autophagy marker protein PfATG8 coinciding with CQR(CC). Taken together, the data show that an unusual autophagy-like process is either activated or inhibited for intraerythrocytic trophozoite parasites at LD50 doses (but not IC50 doses) of CQ, that the pathway is altered in CQR P. falciparum, and that it may contribute along with mutations in PfCRT to confer the CQR(CC) phenotype.
Digital Object Identifier (DOI)
Gaviria, David; Paguio, Michelle F.; Turnbull, Lindsey B.; Tan, Asako; Siriwardana, Amila; Ghosh, Debasish; Ferdig, Michael T.; Sinai, Anthony P.; and Roepe, Paul D., "A Process Similar to Autophagy is Associated with Cytocidal Chloroquine Resistance in Plasmodium Falciparum" (2013). Microbiology, Immunology, and Molecular Genetics Faculty Publications. 19.
Late trophozoite/early schizont stained with anti-ATG8 (green) and anti-apicoplast (red) antibodies. Some overlap between ATG8 and apicoplast localized ACP (D) suggests partial (but not complete, ~25%) co – localization of PfATG8 and apicoplast as previously suggested . Scale bar = 5 µm.
Figure_S2.doc (817 kB)
CQS (HB3, A) and CQR (Dd2, B) parasites treated with IC50 (top row each panel) and 2× IC50 (bottom row) doses of CQ (10, 20 nM and 125, 250 nM, respectively) for 48 hr. Parasites were then stained for ATG8 (green) and with DAPI (blue), as described in Methods. Very few peripherally disposed PfATG8 puncta are observed, supporting the conclusion that puncta formation is associated with LD50, but not IC50 doses of CQ. Scale bar = 5 µm.
Scheme_S1.doc (51 kB)
Cartoon representation of 3D puncta quantification method used in the present work. Hemozoin density (red “x”) is used as the origin, and distances toPfATG8 positive puncta are defined for the reconstructed confocal z stack of images, relative to hemozoin, using 3D Cartesian (x,y,z) coordinates. The cartoon shows an abbreviated depiction of 3 SDCM “slices”, but as described in methods the z stack data set for each cell is a fully assembled, iteratively deconvolved 3D image constructed from approximately 15–20 z slices (see  for additional detail).
Table_S1.doc (48 kB)
LD50 values for the HB3×Dd2 cross progeny. doi:10.1371/journal.pone.0079059.s003
Table_S2.doc (127 kB)
All genes within the chr6 LD50 locus.
Table_S3.doc (97 kB)
All genes within the LD50 chr6×chr8 interaction.
Table_S4.doc (28 kB)
GO enriched molecular functions for the chr6 LD50 locus.
Table_S5.doc (28 kB)
GO enriched biological processes for the LD50 chr6 locus.
Table_S6.doc (30 kB)
GO enriched molecular functions for the LD50 chr6×chr8 interaction.
Table_S7.doc (28 kB)
GO enriched biological processes for the LD50 chr6×chr8 interaction.