An efficient cumate-inducible system for procyclic and bloodstream form Trypanosoma brucei

Li, Feng‐Jun; Xu, Zhi-Shen; Aye, Htay Mon; Brasseur, Anaïs; Lun, Zhao‐Rong; Tan, Kevin S. W.; He, Cynthia Y.

doi:10.1016/j.molbiopara.2017.04.007

Cited by 15 publications

(19 citation statements)

References 21 publications

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“…We reasoned that excess amounts of D/D domains alone might compete with TbArl13 for docking sites, serving as an inhibitor of TbArl13 docking. To test this hypothesis, we overexpressed D/D domain (mCherry–DD) using a cumate (Cmt)-inducible system ( Li et al, 2017 ) in cells expressing endogenously tagged TbArl13–mNeonGreen. In detergent-extracted cells, we consistently observed weaker flagellar mNeonGreen ( Fig.…”

Section: Resultsmentioning

confidence: 99%

The unusual flagellar-targeting mechanism and functions of the trypanosome ortholog of the ciliary GTPase Arl13b

Zhang

Huang

Srivathsan

et al. 2018

Journal of Cell Science

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The small GTPase Arl13b is one of the most conserved and ancient ciliary proteins. In human and animals, Arl13b is primarily associated with the ciliary membrane, where it acts as a guanine-nucleotide-exchange factor (GEF) for Arl3 and is implicated in a variety of ciliary and cellular functions. We have identified and characterized Trypanosoma brucei (Tb)Arl13, the sole Arl13b homolog in this evolutionarily divergent, protozoan parasite. TbArl13 has conserved flagellar functions and exhibits catalytic activity towards two different TbArl3 homologs. However, TbArl13 is distinctly associated with the axoneme through a dimerization/docking (D/D) domain. Replacing the D/D domain with a sequence encoding a flagellar membrane protein created a viable alternative to the wild-type TbArl13 in our RNA interference (RNAi)-based rescue assay. Therefore, flagellar enrichment is crucial for TbArl13, but mechanisms to achieve this could be flexible. Our findings thus extend the understanding of the roles of Arl13b and Arl13b–Arl3 pathway in a divergent flagellate of medical importance..

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Section: Resultsmentioning

confidence: 99%

The unusual flagellar-targeting mechanism and functions of the trypanosome ortholog of the ciliary GTPase Arl13b

Zhang

Huang

Srivathsan

et al. 2018

Journal of Cell Science

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“…Cultivation and differentiation of some life-cycle stages can be achieved in vitro, making T. brucei an ideal organism to study the signaling and cellular remodeling mechanisms during life-cycle differentiation and development. Advanced reverse genetics methods such as inducible expression [3,4] and RNAi [5] allow rapid characterization of protein functions. Additionally, T. brucei possesses a variety of organelles, ubiquitously found in all eukaryotes or uniquely present in trypanosomes, many of them present at single copy and occupy a distinct location in the highly polarized cell.…”

Section: Advantages Of T Brucei As a Model Organism For Cell Biologymentioning

confidence: 99%

Autophagy in Protozoan Parasites: Trypanosoma Brucei as a Model

2017

Future Microbiol.

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" To gain further and more complete understanding of the mechanisms and functions of autophagy, both conserved and evolutionarily divergent, study of more divergent model systems is needed. " Autophagy is an intracellular pathway that clears macromolecules, organelles and intracellular pathogens through autophagosomes and lysosomes upon nutrient limitation, infections and other physiological/pathological conditions. Autophagy has been best studied in budding yeast and mammals, revealing highly conserved machineries and physiological importance in development and diseases. In general, autophagy is triggered upon inhibition of TOR, or activation of AMPK; both conserved protein kinases. Phosphorylation and activation of ATG13 lead to activation of ULK1/2 (Atg1 in yeast), which further stimulates autophagic modules including the PtdIns3K complex: VPS34 and Beclin1, and the unbiquition-like conjugation system that includes ATG8 and ATG12. VPS34 produces PtdIns3P at certain cellular locations, which serves as a signal to recruit other proteins for phagophore formation. The phagophores elongate and engulf cytoplasmic components, forming double-membraned autophagosomes. Finally, autophagosomes are transported to and fused with lysosomes, where the engulfed materials are degraded.Most of the autophagy-related genes identified are highly conserved functionally in yeast and mammals, although different regulatory or signaling mechanisms have been found for some molecules, mTOR as an example [1]. The protozoan parasites are evolutionarily divergent, single-celled eukaryotic pathogens including the Plasmodium spp. that causes malaria; Toxoplasma gondii that causes toxoplasmosis; and Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. that cause the tritryp diseases. Together these pathogens cause huge economic and disease burdens globally. Bioinformatics analysis identified only about half of the ATG homologs in these protozoan parasites [2], implying a simple or divergent autophagy pathway in these organisms. To gain further and more complete understanding of the mechanisms and functions of autophagy, both conserved and evolutionarily divergent, study of more divergent model systems is needed. Advantages of T. brucei as a model organism for cell biology studiesAmong the protozoan parasites, T. brucei is one of the most genetically accessible for study in laboratories. Like many parasites, T. brucei undergoes a complex life cycle in the insect vector and mammalian hosts, taking on different developmental forms. Cultivation and differentiation of some life-cycle stages can be achieved in vitro, making T. brucei an ideal organism to study the signaling and cellular remodeling mechanisms during life-cycle differentiation and development. Advanced reverse genetics methods such as inducible expression [3,4] and RNAi [5] allow rapid characterization of protein functions. Additionally, T. brucei possesses a variety of organelles, ubiquitously found in all eukaryotes or uniquely present in trypanosomes, many of them present at ...

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“…), vanillic acid (Sunter ), and cumate (Li et al. ) switch systems have also been reported for inducible expression of tRNA (Tschopp et al. ) or proteins (Li et al.…”

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confidence: 99%

“…) or proteins (Li et al. ; Sunter ) in T. brucei, although only the vanillic acid system (Sunter ) was appropriate for inducible RNAi.…”

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confidence: 99%

“…To overcome this limitation a tetracycline-regulated expression of dsRNA was established and T. brucei strains stably expressing the tet repressor and T7 RNA polymerase are used for this purpose (Wirtz and Clayton 1995;Wirtz et al 1998). Strains expressing isopropyl b-D-1-thiogalactopyranoside (IPTG) (Tschopp et al 2011), vanillic acid (Sunter 2016), and cumate (Li et al 2017) switch systems have also been reported for inducible expression of tRNA (Tschopp et al 2011) or proteins (Li et al 2017;Sunter 2016) in T. brucei, although only the vanillic acid system (Sunter 2016) was appropriate for inducible RNAi.…”

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confidence: 99%

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A Riboswitch‐based Inducible Gene Expression System for Trypanosoma brucei

Cruz‐Bustos

Ramakrishnan

Cordeiro

et al. 2018

J Eukaryotic Microbiology

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Generation of conditional mutants in Trypanosoma brucei can be done by the use of RNA interference (RNAi). However, RNAi frequently produces off target effects. Here, we present an alternative strategy in which the glmS ribozyme is inserted in the C-terminal region of one allele of a GOI and effectively knocks it down in response to the presence of glucosamine in the culture medium. Using several endogenous genes, we show that the glmS ribozyme cleaves the mRNA in vivo leading to reduction in mRNA and protein expression following glucosamine treatment in both T. brucei procyclic and bloodstream forms. Glucosamine-induced ribozyme activation can be rapidly reversed by removing the inducer. In summary, the glmS ribozyme could be used as a tool to study essential genes in T. brucei.

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An efficient cumate-inducible system for procyclic and bloodstream form Trypanosoma brucei

Cited by 15 publications

References 21 publications

The unusual flagellar-targeting mechanism and functions of the trypanosome ortholog of the ciliary GTPase Arl13b

The unusual flagellar-targeting mechanism and functions of the trypanosome ortholog of the ciliary GTPase Arl13b

Autophagy in Protozoan Parasites: Trypanosoma Brucei as a Model

A Riboswitch‐based Inducible Gene Expression System for Trypanosoma brucei

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