Highlights d Mutations in PfAcAS confer resistance to antiplasmodials MMV019721 and MMV084978 d MMV019721 and MMV084978 specifically inhibit PfAcAS by competing with substrates d cKD and IFA show PfAcAS is an essential nuclear enzyme in blood-stage parasites d PfAcAS inhibitors deplete parasite acetyl-CoA and result in histone hypoacetylation
Understanding the interplay between intracellular pathogens and their hosts is vital to the development of new treatments and preventive strategies. The intracellular “amastigote” stage of the Chagas disease parasite,
Trypanosoma cruzi
, is a critical but understudied parasitic life stage.
In addition to scavenging exogenous cholesterol, the parasitic kinetoplastid Trypanosoma cruzi can endogenously synthesize sterols. Similar to fungal species, T. cruzi synthesizes ergostane type sterols and is sensitive to a class of azole inhibitors of ergosterol biosynthesis that target the enzyme lanosterol 14α-demethylase (CYP51). In the related kinetoplastid parasite Leishmania donovani, CYP51 is essential, yet in Leishmania major, the cognate enzyme is dispensable for growth; but not heat resistance. The essentiality of CYP51 and the specific role of ergostane-type sterol products in T. cruzi has not been established. To better understand the importance of this pathway, we have disrupted the CYP51 gene in T. cruzi epimastigotes (ΔCYP51). Disruption of CYP51 leads to accumulation of 14-methylated sterols and a concurrent absence of the final sterol product ergosterol. While ΔCYP51 epimastigotes have slowed proliferation compared to wild type parasites, the enzyme is not required for growth; however, ΔCYP51 epimastigotes exhibit sensitivity to elevated temperature, an elevated mitochondrial membrane potential and fail to establish growth as intracellular amastigotes in vitro. Further genetic disruption of squalene epoxidase (ΔSQLE) results in the absence of all endogenous sterols and sterol auxotrophy, yet failed to rescue tolerance to stress in ΔCYP51 parasites, suggesting the loss of ergosterol and not accumulation of 14-methylated sterols modulates stress tolerance.
Trypanosoma cruzi
is a protozoan parasite that causes Chagas disease, which causes substantial morbidity and mortality in South and Central America. Throughout its life cycle,
T. cruzi
interacts with insect and mammalian hosts via its single flagellum, establishing intimate contact with host membranes.
The flagellated kinetoplastid protozoan and causative agent of human Chagas disease,Trypanosoma cruzi, inhabits both invertebrate and mammalian hosts over the course of its complex life cycle. In these disparate environments,T. cruziuses its single flagellum to propel motile life stages and in some instances, to establish intimate contact with the host. Beyond its role in motility, the functional capabilities of theT. cruziflagellum have not been defined. Moreover, the lack of proteomic information for this organelle, in any parasite life stage, has limited functional investigation. In this study, we employed a proximity-dependent biotinylation approach based on the differential targeting of the biotin ligase, TurboID, to the flagellum or cytosol in replicative stages ofT. cruzi, to identify flagellar-enriched proteins by mass spectrometry. Proteomic analysis of the resulting biotinylated protein fractions yielded 218 candidate flagellar proteins inT. cruziepimastigotes (insect stage) and 99 proteins in intracellular amastigotes (mammalian stage). Forty of these flagellar-enriched proteins were common to both parasite life stages and included orthologs of known flagellar proteins in other trypanosomatid species, proteins specific to theT. cruzilineage and hypothetical proteins. With the validation of flagellar localization for several of the identified candidates, our results demonstrate that TurboID-based proximity proteomics is an effective tool for probing subcellular compartments inT. cruzi. The proteomic datasets generated in this work offer a valuable resource to facilitate functional investigation of the understudiedT. cruziflagellum.
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