Biology subjects

Burleigh, B. A.

Publications and source records attributed to Burleigh, B. A..

4 recordsLinked to original sources

bioRxiv2023

Proximity-dependent biotinylation and identification of flagellar proteins in Trypanosoma cruzi

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. cruzi uses 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 the T. cruzi flagellum 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 of T. cruzi, to identify flagellar-enriched proteins by mass spectrometry. Proteomic analysis of the resulting biotinylated protein fractions yielded 218 candidate flagellar proteins in T. cruzi epimastigotes (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 the T. cruzi lineage 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 in T. cruzi. The proteomic datasets generated in this work offer a valuable resource to facilitate functional investigation of the understudied T. cruzi flagellum. ImportanceTrypanosoma cruzi is a protozoan parasite that causes Chagas disease, which contributes 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. Currently, few flagellar proteins have been identified in T. cruzi that could provide insight into the mechanisms involved in mediating physical and biochemical interactions with the host. Here, we set out to identify flagellar proteins in the main replicative stages of T. cruzi using a proximity-labeling approach coupled with mass spectrometry. The >200 candidate flagellar proteins identified represent the first large scale identification of candidate flagellar proteins in T. cruzi with preliminary validation. These data offer new avenues to investigate the biology of T. cruzi - host interactions, a promising area for development of new strategies aimed at the control of this pathogen.

microbiology↗

bioRxiv2022

The intracellular amastigote of Trypanosoma cruzi maintains an actively beating flagellum

Throughout its complex life cycle, the uniflagellate parasitic protist, Trypanosoma cruzi, adapts to different host environments by transitioning between elongated motile extracellular forms and non-motile intracellular amastigote forms that replicate in the cytoplasm of mammalian host cells. Despite their name, intracellular T. cruzi amastigotes retain a short flagellum that extends beyond the opening of the flagellar pocket with access to the extracellular milieu. Contrary to the long-held view that the T. cruzi amastigote flagellum is inert, we now report that this organelle is motile and displays quasiperiodic beating inside mammalian host cells. Kymograph analysis determined an average flagellar beat frequency of ~0.7 Hz for intracellular amastigotes. Similar beat frequencies were measured in extracellular amastigotes following their isolation from host cells. Inhibitor studies reveal roles for parasite mitochondrial respiration and intracellular calcium availability in modulating flagellar beat in T. cruzi amastigotes. Together, these findings demonstrate that flagellar motility is an intrinsic property of T. cruzi amastigotes and suggest that this organelle may play an active role in the parasite infection process. To our knowledge, this is the first record of an intracellular eukaryotic flagellum beating within another eukaryotic cell.

cell biology↗

bioRxiv2022

Fatty acid elongases 1-3 have distinct roles in mitochondrial function, growth and lipid homeostasis in Trypanosoma cruzi.

Trypanosomatids are a diverse group of uniflagellate protozoa that include globally important pathogens such as Trypanosoma cruzi, the causative agent of Chagas disease. Trypanosomes lack the fatty acid synthase (FAS)-I system typically used for de novo synthesis of long chain fatty acids (LCFA) in other eukaryotes. Instead, these microbes have evolved a modular fatty acid elongase (ELO) system comprised of individual ELO enzymes that operate processively. The role of the ELO system in maintaining lipid homeostasis in trypanosomes has not been determined. Here we demonstrate that ELO2 and ELO3 are required for global lipidome maintenance in the insect stage of T. cruzi whereas ELO1 is dispensable for this function. Instead, ELO1 activity is needed to sustain mitochondrial activity and normal growth. The cross-talk between microsomal ELO1 and the mitochondrion is a novel finding that merits examination of the trypanosomatid ELO pathway as critical for central metabolism.

microbiology↗

bioRxiv2022

Endogenous sterol synthesis is dispensable for Trypanosoma cruzi epimastigote growth but not stress tolerance

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 14-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 ({Delta}CYP51). Disruption of CYP51 leads to accumulation of 14-methylated sterols and a concurrent absence of the final sterol product ergosterol. While{Delta} CYP51 epimastigotes have slowed proliferation compared to wild type parasites, the enzyme is not required for growth; however,{Delta} CYP51 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 ({Delta}SQLE) results in the absence of all endogenous sterols and sterol auxotrophy, yet failed to rescue tolerance to stress in{Delta} CYP51 parasites, suggesting the loss of ergosterol and not accumulation of 14-methylated sterols modulates stress tolerance.

microbiology↗