Date of Award
12-2022
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Microbiology
Committee Chair/Advisor
Joshua Alper
Committee Member
Zhicheng Dou
Committee Member
Kimberly Paul
Committee Member
Lesly Temesvari
Committee Member
James Morris
Abstract
Trypanosoma brucei is a parasitic kinetoplastid that causes African trypanosomiasis and is transmitted to a mammalian host by the tsetse fly (Glossina spp.). T. brucei relies on its flagellar motility to carry out its morphological changes from the procyclic form (predominant in the fly vector) to the bloodstream form (infectious form in mammals) and navigate the bloodstream of its host. The driving structure within the flagellum is the axoneme, which is composed of microtubules and dynein motor proteins. The tubulin code hypothesis suggests that cells regulate microtubule motor protein activity through post-translational modifications (PTMs) of alpha and beta tubulin. However, the regulatory role of tubulin PTMs on the flagellar beat and structural effects on the subpellicular microtubules that dictate T. brucei’s unique morphology remain largely unknown. To characterize the importance of tubulin PTMs, I targeted the enzymes responsible for tubulin acetylation (alpha-tubulin acetyltransferase 1) and detyrosination (vasohibin) using two different strategies. First, we generated knockdowns of both enzymes through a well-developed RNAi system using the pZJM vector which revealed a >50% decrease in transcript and PTM presence. Second, we modified a CRISPR/Cas9 system to knock out both enzymes to compare gene expression and modification presence between cell lines. RT-qPCR and western blot analysis revealed complete absence of PTM enzyme transcripts and PTM presence in whole cell lysate. Confocal immunofluorescence revealed the subpellicular microtubules are highly acetylated and detyrosinated. Cell tracking and sedimentation assays revealed a significant increase in motility and swimming speed in non-acetylated cell lines. However, cells containing heavily tyrosinated microtubules displayed an insignificant motility decrease in comparison to wild type. These results indicate acetylation negatively-regulates the dynein-driven flagellar motility of T. brucei, whereas detyrosination as little to no effect. Additionally, the subpellicular-microtubule array is significantly disrupted in the absence of acetylated alpha tubulin, leading to an elongated trypanosome (>14 μm). Overall, the findings from this study support the evolutionary conservation of tubulin PTMs in simple eukaryotes, such as T. brucei, and thus, can be expanded to similar Leishmania and Trypanosoma species.
Recommended Citation
Wentworth, Katherine, "The effects of tubulin post-translational modifications on the flagellar motility of Trypanosoma brucei" (2022). All Theses. 3948.
https://tigerprints.clemson.edu/all_theses/3948
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