Figures and data
CIP demonstrates potent inhibition on Babesia spp..
(A) and (B) Dose-dependent growth curve of CIP on B. bovis and B. gibsoni in vitro. Each value represents the mean ± standard deviation (SD) of three independent experiments carried out in triplicate. (C) Inhibitory effects of CIP and atovaquone (ATO) plus azithromycin (AZI) on the proliferation of B. microti in BALB/c mice. (D) Hematocrit (HCT) values in mice treated with CIP or ATO plus AZI compared with vehicle-treated mice. (E) Inhibitory effects of CIP and atovaquone (ATO) plus azithromycin (AZI) on the proliferation of B. rodhaini in BALB/c mice. (F) Survival rates of CIP-treated, ATO plus AZI-treated, and vehicle-treated mice. The treatment time is shown by arrows, and significant differences (P < 0.01) between the drug-treated groups and the vehicle-treated control group are indicated by asterisks. The data from one of six individual experiments are expressed as means ± SD. *, P ˂ 0.05; **, P ˂ 0.01.
Mutations in BgATP4 mediate CIP resistance.
(A-C) Representative sequencing chromatogram of resistant parasites from CIP-treated B. gibsoni. The resistant parasite genomic DNA is extracted from blood samples after a 60 day-treatment. The BgATP4 gene was amplified and sequenced using the DNA. (D) Dose-dependent growth curve of BgATP4WT, BgATP4L921V, and BgATP4L921I in vitro. Each value represents the mean ± standard deviation (SD) of three independent experiments carried out in triplicate.
Effect of CIP on [Na+]i and pH regulation in B. gibsoni lines.
(A) Untreated and CIP-treated parasite morphology after incubation for 72 h. Scale bar: 5 µm. (B) Sizes of 100 parasites in two groups measured with ImageJ software in panels A. Statistically significant differences between the means of variables determined by t-test. ****, P ˂ 0.0001. (C) [Na+]i concentrations after addition of CIP in BgATP4WT line. Representative traces from the experiment that highlight the impact of adding 20 nM CIP (green), 1 nM CIP (blue), or 0 nM CIP (grey) on the concentration [Na+]i of the BgATP4WT line. (D) Alkalinization of pHi in BgATP4WT line upon addition of the ATP4 inhibitor. (E) Addition of 20 nM CIP to the wild-type and resistant parasite lines results in different [Na+]i concentrations. (F) Addition of 20 nM CIP to the wild-type and resistant parasite lines results in different pHi concentrations. Experiments were performed in technical duplicates for at least three biological repeats. ns, non significant, p ˃ 0.05; *, P ˂ 0.05; **, P ˂ 0.01.
Binding sites proximal to BgATP4 residue 921 predicted by molecular docking.
(A) The lowest energy poses for CIP were located in reference to the whole protein structure, docking against the WT (green), L921V (yellow), and L921I (pink) mutant BgATP4. The side chain of L921 is also shown in a red stick at its position. (B-D) The zoomed views of the binding locations of CIP.
Cytotoxicity assay of CIP on the MDCK cell line.
The MTP-500 microplate reader is utilized to detect the absorbance at 450 nm. The results are displayed as the mean ± standard deviation of three separate tests.
Multiple sequence alignment of ATP4 in different species.
A yellow square denotes the BgATP4 mutation site discovered in this investigation; red squares represent sites linked to P. falciparum CIP resistance, and a green square represents sites associated with T. gondii.
Binding sites for CIP found by Gnina search across the entire surface of the protein.
(A-C) The binding space of WT, L921V, and L921I mutants in BgATP4 are labeled in green, yellow and pink, respectively.
Proposed mechanism of inhibition of CIP on wild-type and mutant parasite-infected erythrocytes.
CIP disrupts the BgATP4 function of wild-type parasites, which causes a net influx of Na+ and efflux of H+ from the parasite. The osmotic load imposed on the influx of Na+ further brings about parasite swelling and internal alkalinization, which are the main factors in Babesia death. Mutations in ATP4 minimize the susceptibility to ATP4 inhibitors by recovering H+ and Na+ balance.
Primer sets of B. gibsoni ATP4
