Characterising kinetoplastid pantothenate kinase, a multifunctional enzyme of the CoA biosynthesis pathway

Kinetoplastid parasites of the Trypanosoma and Leishmania genera cause fatal diseases of the tropics, subtropics and Mediterranean basin, in the form of Human African Trypanosomiasis, Chagas disease and Leishmaniasis. In addition to debilitating effects on the health of thousands of individuals, these illnesses have a crippling impact on the global economy, with an estimated annual cost of over 7 billion US dollars. Despite their clinical and economic importance, efforts to develop safe and effective treatments have made limited progress since discovery of these pathogens over 100 years ago. Understanding parasite biochemistry is critical to the development of effective therapies. Coenzyme A (CoA) is a cofactor required for hundreds of metabolic reactions and its biosynthesis pathway may be a target for drugs against apicomplexa, fungi and bacteria. However, in kinetoplastids this pathway has been poorly characterised. The first step in CoA synthesis requires pantothenate kinase (PanK). In this work, PanK is shown to be structurally unique in kinetoplastids compared to other eukaryotes as it contains two additional fused domains, putatively identified by homology as conferring phosphodiesterase and adenylating activity. By a combination of genetic and biochemical approaches, we investigated the role of PanK in growth and survival of the pathogens Trypanosoma cruzi and T. brucei. Tagging of T. cruzi PanK by CRISPR spCas9 revealed expression of an extra-nuclear multi-domain polypeptide (~170kDa). Gene deletion using CRISPR was not achievable in T. cruzi epimastigotes, suggesting that at least one activity of the protein is essential to this life cycle stage. Similarly, PanK depletion in bloodstream-form T. brucei by RNAi was lethal, a phenotype that was partially rescued by CoA supplementation. Add-back experiments revealed that constitutive expression of the T. cruzi orthologue rescues RNAi lethality in T. brucei, demonstrating functional homology of the two enzymes. Furthermore, using the same method we found that the additional fused domains are critical to function, as the isolated T. cruzi PanK domain does not rescue lethality of T. brucei PanK depletion. This is in spite of the fact that this domain is active, as demonstrated by kinase assays. We also found that within the PanK domain, Arg-1270 is critical to function and important for the in vitro proliferation of T. brucei. The putative phosphodiesterase appears not to play an essential role in proliferation of this species, at least when the adenylating and PanK domains are active. We conclude that CoA synthesis is essential for the in vitro growth of clinically important trypanosomes. The finding of a kinetoplastid-specific multi-functional PanK raises the possibility of therapeutic exploitability of this step of the biosynthesis pathway in kinetoplastids.