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. 2017 Aug 18;6:e29865. doi: 10.7554/eLife.29865

Figure 3. Knockdown of ftsH1 in P. falciparum leads to apicoplast loss and hypersensitivity to actinonin.

(a) Schematic of the endogenous knockdown strategy. When aTC is present in the media, the tet-repressor binds aTC and does not bind the 10x-aptamer sequence, which relieves translational repression, allowing PfFtsH1 to be expressed. When aTC is washed out of the media, the tet-repressor binds the 10x-aptamer and prevents expression of PfFtsH1. (b) Time course of parasite growth without aTC and in the presence or absence of IPP in the media, normalized to the untreated or IPP-rescued parental strain as appropriate. Error bars represent the SEM of two biological replicates. (c) Time course of the apicoplast:nuclear genome ratio measured by quantitative PCR (qPCR) using primers for the apicoplast and nuclear genomes during treatment with or without aTC. All samples contained IPP to rescue parasite growth. Genome ratios were normalized to respective parental cultures also grown with IPP. Error bars as in c. (d) Dose-dependent parasite growth inhibition by actinonin in the absence or presence of aTC. Error bars as in c.

Figure 3—source data 1. Oligonucleotide primers and plasmids used in this study.
elife-29865-fig3-data1.xlsx (53.1KB, xlsx)
DOI: 10.7554/eLife.29865.015
Figure 3—source data 2. Numerical data for Figure 3 and Figure 3—figure supplement 3 .
elife-29865-fig3-data2.xlsx (48.7KB, xlsx)
DOI: 10.7554/eLife.29865.016

Figure 3.

Figure 3—figure supplement 1. C-terminal cleavage of PfFtsH1 is dependent on the presence of the apicoplast and efficiency of PfFtsH1 knockdown can be assessed in parasites missing their apicoplast.

Figure 3—figure supplement 1.

(A) Western blot of PfFtsH-FLAG levels in parasites with and without an apicoplast and with and without aTC induction. Consistent with the previous report of PfFtsH1 C-terminal processing, we were unable to detect full-length PfFtsH1-FLAG using anti-FLAG in parasites with intact apicoplasts (lane 1) (Tanveer et al., 2013; Karnataki et al., 2009). However, in parasites missing apicoplasts, full-length PfFtsH1-FLAG was detectable, suggesting that PfFtsH1 processing requires the apicoplast (lanes 2). PfFtsH1-FLAG is not observed in parasites with or without apicoplasts if there is no aTC induction, indicating that the band representing FtsH-FLAG is specific and not an artifact of missing apicoplasts (lanes 3–4 respectively). All samples contain IPP to rescue growth and Cas9-FLAG is used as a loading control. Each sample was taken at the trophozoite stage. (B) To assess the knockdown efficiency of PfFtsH1, we used a western blot comparing PfFtsH1-FLAG levels in the presence (lanes 1–4) or absence of aTC (lanes 5–8) in IPP-rescued parasites missing their apicoplast. Each sample was taken at the trophozoite stage and cycle 0 indicates 24 hr after the removal of aTC. Lanes 9 and 10 are samples from the parental strain that do not contain the FLAG-tag or the aptamer sequence in the 3’ UTR of PfFtsH1. In each case, Cas9-FLAG was used as a loading control. PfFtsH1-FLAG levels were reduced to undetectable levels at 24 hr after aTC removal, validating our knockdown strategy.
Figure 3—figure supplement 2. Knockdown of PfFtsH1 specifically disrupts the apicoplast and leads to specific hypersensitivity to actinonin.

Figure 3—figure supplement 2.

(a) Time course of parasite growth with or without aTC and with or without IPP in the media. IPP rescues the growth defect observed in upon PfFtsH1 downregulation, indicating that PfFtsH1 is essential for an apicoplast-specific function. Growth is shown normalized to the untreated or IPP-rescued parental strain as appropriate. Error bars represent the SEM of two biological replicates. (b) Dose-dependent parasite growth inhibition by actinonin with or without aTC for the parental (red) and PfFtsH (black) strain. The EC50 of the parental strain is unchanged by the removal of aTC. Error bars as in a. (c) Dose-dependent parasite growth inhibition by fosmidomycin with or without aTC and with or without IPP. The fosmidomycin EC50 is unchanged by regulating levels of PfFtsH1, indicating that the observed hypersensitivity to actinonin upon knockdown of PfFtsH1 is specific to actinonin and does not occur for all apicoplast drug. Error bars represent the SEM of three technical replicates. (d) Dose-dependent parasites growth inhibition by chloramphenicol during the second lytic cycle (120 hr) with or without aTC. Error bars represent the SEM of three technical replicates. The chloramphenicol EC50 is unchanged by regulating levels of PfFtsH1, indicating that the observed hypersensitivity to actinonin upon knockdown of PfFtsH1 is specific to actinonin and does not occur for all apicoplast drugs.