Venturicidin A affects the mitochondrial membrane potential and induces kDNA loss in Trypanosoma brucei

ABSTRACT Neglected tropical diseases caused by trypanosomatid parasites have devastating health and economic consequences, especially in tropical areas. New drugs or new combination therapies to fight these parasites are urgently needed. Venturicidin A, a macrolide extracted from Streptomyces, inhibits the ATP synthase complex of fungi and bacteria. However, its effect on trypanosomatids is not fully understood. In this study, we tested venturicidin A on a panel of trypanosomatid parasites using Alamar Blue assays and found it to be highly active against Trypanosoma brucei and Leishmania donovani, but much less so against Trypanosoma evansi. Using fluorescence microscopy, we observed a rapid loss of the mitochondrial membrane potential in T. brucei bloodstream forms upon venturicidin A treatment. Additionally, we report the loss of mitochondrial DNA in approximately 40%–50% of the treated parasites. We conclude that venturicidin A targets the ATP synthase of T. brucei, and we suggest that this macrolide could be a candidate for anti-trypanosomatid drug repurposing, drug combinations, or medicinal chemistry programs.

distinct nature of the trypanosomal ATP synthase complex.The ATP synthase complex in T. brucei is composed of at least 25 subunits (4, 10-13), 14 of which are specific to and conserved among kinetoplastids (10).
Apart from amphotericin B, other macrolide compounds have shown activity against trypanosomatid parasites (24)(25)(26).Among these are known inhibitors of the ATP synthase of fungi and bacteria (27,28), for example, venturicidin A (Fig. 1).Given venturicidin A's activity against T. b. brucei (24), we were interested in determining its potential against other trypanosomatid parasites.We further used T. b. brucei as a model to characterize the effect that venturicidin A treatment has on trypanosomatid parasites.

RESULTS
Venturicidin A (Fig. 1) was found to have a selective antitrypanosomal activity in vitro (24).We evaluated the in vitro efficacy of venturicidin A against bloodstream form T. b. brucei 2T1, T. b. rhodesiense, Trypanosoma evansi, as well as promastigote and amasti gote forms of Leishmania donovani using the Alamar Blue assay (30,31).Venturicidin A showed a 50% inhibitory concentration (IC 50 ) against T. b. brucei 2T1 of 21.49 nM (Fig. 1) and an IC 50 value of 5 nM against T. b. rhodesiense (Fig. 1).Both values are slightly lower than the ones published previously, which were 160 nM for T. b. brucei and 720 nM for T. b. rhodesiense (24).
Here, we report for the first time that venturicidin A acts as a potent inhibitor of the human pathogen Leishmania donovani, exerting activity against both the promastigote and amastigote forms.We further show that Trypanosoma evansi is less susceptible to venturicidin A than T. b. brucei.
Venturicidin A is known to act on the F 0 F 1 -ATP synthase (5,28,32,33).To further investigate its effect on T. b. brucei, we treated T. b. brucei 2T1 bloodstream forms with 1× IC 50 (21 nM), 5× IC 50 (107 nM), or 8× IC 50 (172 nM) venturicidin A for 24 h and assessed the mitochondrial membrane potential.The treated cells, together with untreated cells as controls, were stained with MitoTracker for 30 min in culture, then fixed and placed onto microscopy slides.
As expected, we observed a clear MitoTracker fluorescence signal in untreated cells, indicative of an intact mitochondrial membrane potential (Fig. 2).In venturicidin A treated cells, independent of the drug concentration used, no MitoTracker signal was visible, indicating the collapse of the mitochondrial membrane potential (Fig. 2).
Additionally, we noticed that a consistent portion of cells had no detectable mitochondrial DNA, also known as kinetoplast DNA (kDNA) (Fig. 2).To confirm this observation, we treated the parasites with 1× IC 50 (21 nM), 5× IC 50 (107 nM), or 8× IC 50 (172 nM) venturicidin A for 24 h, fixed the cells and stained them with DAPI to mark the nucleus (N) and the kDNA (K), and with an anti-α-tubulin antibody to visualize the cell shape (Fig. 3A).Within a normal population (with kDNA = 1K1N, 2K1N, 2K2N), the majority of the cells, around 75%-80%, are in the G 1 phase (1K1N) (34).The percentage of cells that have completed the S-phase and therefore have duplicated both the kDNA and the nucleus (2K2N) and are entering cytokinesis is less than 10% (34).Compared to the untreated cells, around 40% of the cells treated with either 1× or 5× IC 50 venturicidin A had lost their kDNA after 24 h, whereas with 8× IC 50 venturicidin A treatment, the percentage of cells without a detectable kDNA reached up to 50% (without kDNA = 0K1N) (Fig. 3B).Under all venturicidin A concentrations tested, we also observed dividing cells with an abnormal number of kDNA, such as one kinetoplast but two nuclei (wrong # kDNA = 1K2N) (Fig. 3A), in spite of the fact that in the cell cycle of T. b. brucei, the kinetoplast divides before the nucleus (35).This possibly indicates how the cells without a detectable kDNA could have originated: a cell with 1K2N divides into a cell with 1K1N and one with 0K1N.
Our results are in agreement with an action of venturicidin A on the ATP synthase resulting in the loss of the mitochondrial membrane potential, and they show for the first time an effect of venturicidin A on the kDNA.

DISCUSSION
The antitrypanosomal activity of venturicidin A has been known for more than a decade (24).The current experiments resulted in a slightly lower IC 50 value for venturicidin A against T. b. brucei compared to the IC 50 value reported previously.Both IC 50 values were determined with the same method (30) but using two different T. b. brucei strains: 2T1 in this study and GUTat 3.1 previously.We further observed that venturicidin A's activity extends to Leishmania donovani, the main causative agent of visceral leishmaniasis, as well as the animal pathogen Trypanosoma evansi.
Our results in T. b. brucei parasites are in agreement with previously published observations regarding the action of venturicidin A on the ATP synthase complex of fungi, bacteria, and trypanosomes (5,28,32,33).In fact, upon venturicidin A treatment, we observed the collapse of the mitochondrial membrane potential, which in T. b. brucei bloodstream forms is maintained by the ATP synthase operating in reverse mode.The same effect has been observed previously in T. b. brucei with the ATP synthase inhibitor oligomycin (5,36).
It is the first time (to our knowledge) that loss of kDNA was observed upon ventur icidin A treatment.kDNA loss has previously been described as a result of treatment with drugs like the DNA intercalating agents phenanthridine (also known as ethidium bromide) or acriflavine (37)(38)(39)(40) but has so far not been reported after treatment with a macrolide.
Trypanosoma evansi and Trypanosoma equiperdum, responsible for Surra and Dourine, respectively, occur in nature also as dyskinetoplastic forms (39).The introduction of a mutation in the gamma-subunit of the mitochondrial ATP synthase, γ-L262P, compen sates for the dyskinetoplasy (absence of kDNA) in T. b. brucei (6,41), without affecting cell viability.In fact, the subsequent loss of gene products encoded in the kDNA is compensated for, by an electrogenic exchange of ADP 3− for ATP 4− via the ATP/ADP carrier, which allows the cells to maintain their mitochondrial membrane potential (41).It is very unlikely that the kDNA loss, observed upon venturicidin A treatment, is the consequence of such a genomic mutation, as the loss of kDNA is already observed within 24 h and because venturicidin A treatment, in contrast to the mutation, indu ces parasite death.The effect of venturicidin A on naturally occurring dyskinetoplastic trypanosomes requires further attention, especially because induced as well as naturally occurring dyskinetoplastic T. evansi have been shown to lose their sensitivity toward the macrolide oligomycin (42).The finding that T. evansi, which does not require an active mitochondrion, is less susceptible to venturicidin A than T. b. brucei is in agreement with the proposed mode of action, supporting the hypothesis that the trypanocidal activity of venturicidin A is due to collapse of the mitochondrial membrane potential.The rise of drug resistance is a threat to global health that is increasingly alarm ing health professionals and scientists.In light of the time-intensive and cost-inten sive process of developing new antimicrobial agents, repurposing of already available drugs can be an efficient alternative.Additionally, combination therapies can delay the emergence of drug resistance, as well as lower the dosage of the individual combina tion partners (43).Both drug repurposing and combination therapies have resulted in new treatment strategies for trypanosomatid infections.A successful example of a combination therapy to treat human African trypanosomiasis is NECT, a combination of nifurtimox and eflornithine (44)(45)(46) that is effectively used to treat second-stage T. b. gambiense infections.One of the most recent examples of successful drug repur posing is fexinidazole, the first orally available treatment against both stages of T. b. gambiense infections (23,47).Venturicidin A is highly active against trypanosomatids and it will be interesting to further study its mode of action in Leishmania subsp.and T. cruzi.In bacteria, combinations of venturicidin A and aminoglycoside antibiotics have been shown to be synergistic (48).We, therefore, suggest to also test venturicidin A in combination with known antitrypanosomal drugs.
Understanding the effects of venturicidin A on the trypanosomal ATP synthase complex will provide valuable insight into the biology of trypanosomatid parasites.The introduction of bedaquiline for the treatment of multidrug-resistant Mycobacterium tuberculosis infections (49) illustrates that the ATP synthase can be successfully targe ted in order to fight microbial infections (CDC Guidelines: https://www.cdc.gov/mmwr/preview/mmwrhtml/rr6209a1.htm).Therefore, we suggest that it is worth studying the ATP synthase complex as a potential drug target in the context of trypanosomatid parasites.
Peritoneal mouse macrophages from CD-1 mice were cultured at 37°C with 5% CO 2 in RPMI 1640 medium with 10% FCS.

Drug assay
Venturicidin A (Santa Cruz sc-202380A) IC 50 values were measured by Alamar Blue assay as described previously in references (30,54), and for intracellular assays as described in reference (31), both with minor adjustments.

For extracellular assay
In a 96-well plate, each well was supplemented with 50 µL (60 µL for axenic amasti gotes L. donovani) culture medium with the corresponding serial drug dilutions.Fifty microliters (60 µL for axenic amastigotes L. donovani) culture medium with parasites was added to each well.After 70 h of incubation, 10 µL (0.125 mg/mL) of resazurin was added to each well, and the plates were incubated for 2 h.For axenic amastigotes L. donovani, plates were incubated for 69 h and 3 h with resazurin, whereas promastigotes L. donovani were incubated for 66 h and 6 h with resazurin.The fluorescence was quantified with a SpectraMax reader (Molecular Devices) and SoftMax Pro 5.4.6 Software, using an excitation wavelength of 536 nm and an emission wavelength of 588 nm.

For intracellular assay
Mouse peritoneal macrophages were seeded into a 96-well plate.After 24 h, 100 µL of amastigote L. donovani was added.After 24 h, the medium containing free amastigote forms was removed and replaced with fresh medium.Serial drug dilution was added to the wells.After 96 h of incubation, the medium was removed, and the cells were fixed with 50 µL of 4% formaldehyde solution followed by staining with a 5-µM DRAQ5 (BioStatus Limited, UK).Plates were imaged in Operetta CLS (PE) microscope using a 20× air objective.Automated image analysis was performed with the Operetta CLS (PE).The IC 50 values were calculated based on the infection rate and the number of intracellular amastigotes.

FIG 3
FIG 3 Venturicidin A treatment induces kDNA loss.(A) Immunofluorescence of the untreated Tb brucei 2T1 cell line, followed by cells treated for 24 h with 1×, 5×, or 8× IC 50 of venturicidin A (VentA).The lower panel for each venturicidin A concentration shows dividing cells.The cells were stained with DAPI (blue) as a DNA marker and an anti-α-tubulin antibody (red) to visualize the cell body.The kinetoplasts (kDNAs), where present, are marked with "k".Scale bar 5 µm.(Continued on next page)