Bumped kinase inhibitor prohibits egression in Babesia bovis

Abstract

Babesiosis is a global zoonotic disease acquired by the bite of a Babesia-infected Ixodes tick or through blood transfusion with clinical relevance affecting humans and animals. In this study, we evaluated a series of small molecule compounds that have previously been shown to target specific apicomplexan enzymes in Plasmodium, Toxoplasma and Cryptosporidium. The compounds, bumped kinase inhibitors (BKIs), have strong therapeutic potential targeting apicomplexa-specific calcium dependent protein kinases (CDPKs). We investigated if BKIs also show inhibitory activities against piroplasms such as Babesia. Using a subset of BKIs that have promising inhibitory activities to Plasmodium and Toxoplasma, we determined that their actions ranged from 100% and no inhibition against Babesia bovis blood stages. One specific BKI, RM-1-152, showed complete inhibition against B. bovis within 48h and was the only BKI that showed noticeable phenotypic changes to the parasites. Focusing our study on this BKI, we further demonstrated that RM-1-152 has Babesia-static activity and involves the prohibition of merozoite egress while replication and re-invasion of host cells are unaffected. The distinct, abnormal phenotype induced by RM-1-152 suggests that this BKI can be used to investigate less studied cellular processes such as egression in piroplasm.

Keywords: Babesia; Bumped kinase inhibitors; Chemotherapy; Egression; Static.

Figure 1

Effect of bumped kinase inhibitors (BKIs) at 30 μM on Babesia asexual erythrocytic stages over 72h. (A) BKIs with partial to no inhibition and (B) BKIs with inhibitory effect as early as 24h post-incubation. NT, no treatment control; ID, imidocarb diproprionate positive control; PE, parasitized erythrocytes.

Figure 2

Visualization of Babesia bovis under the influence of 30 μM RM-1-152 after 24h. (A) No treatment control, showing twin tear-drop shaped merozoites occupying a single erythrocyte. (B-C) Five circular merozoite-like parasites within a single erythrocyte and (D-E) infected erythrocytes harboring >5 circular merozoites per cell under continuous presence of RM-1-152.

Figure 3

Babesia bovis successfully re-invaded uninfected erythrocytes upon the removal of bumped kinase inhibitor’s effect. Merozoites from the RM-1-152-treated (A) and no treatment (NT) (B) groups were freed by erythrocyte lysis. (C) and (D) correspond to merozoites-infected erythrocytes previously exposed to RM-1-152 in the presence (C) and absence (D) of 30 μM RM-1-152 at 72h, respectively while (E) and (F) correspond to NT-merozoites reinvaded erythrocytes in the presence and absence of 30 μM RM-1-152 at 72h, respectively. Comparison of B. bovis-infected erythrocytes using quantitative RT-PCR amplifying (G) BBOV_II004820 and (H) BBOV_II001650 transcripts in four treatment groups at 144h. No treatment, NT control; BKI, RM-1-152 at 30 μM; CL, 20 μM clindamycin hydrochloride and ID, 20 μM imidocarb dipropionate. *, statistically significant with p<.05.