Culturing and Transfection of Pleomorphic Trypanosoma brucei

Bachmaier, Sabine; Thanner, Theresa; Boshart, Michael

doi:10.1007/978-1-0716-0294-2_2

Cited by 9 publications

(14 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Pleomorphic T. brucei parasites (EATRO 1125, AnTat1.1 90:13)[20] were grown in vitro in the presence of BME and the effects on cell proliferation assessed with respect to growth in HMI-9 liquid trypanosome culture medium (Fig 1A). Given the possibility that increasing the viscosity of the medium could induce cells to differentiate, we also tested the ability of cells to differentiate in Geltrex, an extracellular matrix with 10 times less protein concentration than BME but with similar gelling properties, as well as HMI-9 containing agarose 0.65%(w/v) and 1.1%(w/v) methylcellulose, which have been used previously to support pleomorphic cell growth [23, 24]. Cells grew at comparable rates during the first 48 hrs but by 72 hrs parasites in the presence of BME showed a stumpy-like phenotype.…”

Section: Resultsmentioning

confidence: 99%

Basement membrane proteins as a substrate for efficientTrypanosoma bruceidifferentiation in vitro

Rojas

Cayla

Matthews

2021

Preprint

View full text Add to dashboard Cite

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo , we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms . BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro , procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.

show abstract

Section: Resultsmentioning

confidence: 99%

Basement membrane proteins as a substrate for efficientTrypanosoma bruceidifferentiation in vitro

Rojas

Cayla

Matthews

2021

Preprint

View full text Add to dashboard Cite

show abstract

“…The BSF of AnTat1.1 90-13, a pleomorphic T. brucei brucei cell line constitutively expressing T7 RNA polymerase and tetracycline (tet) repressor [62,63], was cultured at 37°C and 5% CO 2 atmosphere in modified HMI-9 medium [64] containing 10% (V/V) heat-inactivated FCS. The procyclic form (PCF) was cultivated at 27°C in modified SDM-79 medium [65] supplemented with 10% (V/V) heat-inactivated FCS, 7.5 mgÁL −1 hemin, 10 mM glucose and 10 mM glycerol.…”

Section: T Brucei Cultivationmentioning

confidence: 99%

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

et al. 2021

Self Cite

View full text Add to dashboard Cite

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE-like domains are present in few eukaryotic taxa, for example the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4-kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

show abstract

“…Pleomorphic T. brucei parasites (EATRO 1125, AnTat1.1 90:13) [19] were grown in vitro in the presence of BME and the effects on cell proliferation assessed with respect to growth in HMI-9 liquid trypanosome culture medium (Fig 1A). Given the possibility that increasing the viscosity of the medium could induce cells to differentiate, we also tested the ability of cells to differentiate in Geltrex, an extracellular matrix with 10 times less protein concentration than BME but with similar gelling properties, as well as HMI-9 containing agarose 0.65%(w/v) and 1.1%(w/v) methylcellulose, which have been used previously to support pleomorphic cell growth [22,23]. Cells grew at comparable rates during the first 48 hrs but by 72 hrs parasites in the presence of BME showed a stumpy-like phenotype.…”

Section: Evaluation Of Basement Membrane Extract Containing Medium For Parasite Growth and Differentiationmentioning

confidence: 99%

Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro

2021

View full text Add to dashboard Cite

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo, we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms. BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro, procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.

show abstract

Culturing and Transfection of Pleomorphic Trypanosoma brucei

Cited by 9 publications

References 45 publications

Basement membrane proteins as a substrate for efficientTrypanosoma bruceidifferentiation in vitro

Basement membrane proteins as a substrate for efficientTrypanosoma bruceidifferentiation in vitro

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro

Contact Info

Product

Resources

About