Characterization of Babesia bovis apicoplast metabolome

Marina Caballero

The multi-membranous apicoplast recently gains attention due to its potential as a chemotherapeutic target. Disruption of the prokaryotic-derived type II fatty acid (FASII) and non-mevalonate 1-deoxy-D-xylulose-5-phosphate (MEP) biosyntheses pathways, found exclusively within the apicoplasts' lumens in related Apicomplexans such as Plasmodium falciparum and Toxoplasma gondii, has proven lethal to the parasites with minimal side effects to the mammalian hosts. Babesia bovis genome sequencing project recently reports the presence of an apicoplast genome. Due to the phylogenetic relatedness of Babesia sp. and Plasmodium sp., FASII and MEP apicoplastic pathways may also be functional in Babesia sp. FASII pathway requires acyl carrier protein (ACP), while MEP pathway requires 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, LytB (IspH) in isoprenoid substrate production. We investigated the presence of Bbacp and BblytB, their transcripts and translated products. In silico characterization suggests the presence of putative N-terminal bipartite signals in both proteins, a prerequisite shared by many nuclear-encoded apicoplast targeted luminal proteins in related Apicomplexans. Antibodies specific to BbACP and BbLytB were generated to localize these proteins in B. bovis. Our results confirm that Bbacp and BblytB are actively transcribed, translated and both proteins are confined to a cellular compartment distinct from the nucleus and mitochondrion. We demonstrate the requirement of the putative bipartite signals in directing protein to the apicoplast lumen. Drug inhibitory studies were carried out to investigate the functionality of both pathways. Similarly found in Plasmodium, we show that MEP pathway likely exists as specific drug inhibition proved lethal to B. bovis. In contrast, multiple drugs targeting FASII substrates failed to inhibit B. bovis growth, suggesting the absence of a conserved FASII pathway. In conclusion, this study reports the existence of MEP pathway within B. bovis which occurs within the apicoplastic lumen where BbLytB actively participates. On the contrary, conserved FASII pathway does not exist and BbACP may function in an alternative pathway in the apicoplast or is a vestigial protein which once served in a now extinct FASII pathway. It is also plausible that if FASII exists within B. bovis, components may be so divergent that established FASII inhibitory drugs fail to target them.

Characterization of Babesia bovis apicoplast metabolome

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