Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Abstract

dIn vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra-and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; ␣ ‫؍‬ 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening.T rypanosoma brucei is the kinetoplastid parasite responsible for human African trypanosomiasis (HAT), a potentially fatal infection of sub-Saharan Africa. Current treatments for HAT are inadequate because of high toxicity and complex administration regimens, and new drugs are urgently required to ensure ongoing control of the disease in the face of emerging reports of drug resistance (1). Newly invigorated efforts to bring novel compounds forward as drugs have led to several promising candidates emerging through screening against parasites in culture (2-5). In vitro continuous culture methods enable the routine screening of compounds for trypanocidal activity against bloodstream form trypanosomes, for example, with resazurin fluorescence-based assays (5-7).Culture methods for bloodstream form T. brucei were first developed in the 1970s and required feeder layers to support continuous growth (8). This requirement could be overcome by regular supplementation with cysteine (9) and supplementation with mammalian serum (10). Continuous culture was successfully maintained by the addition of reducing and chelating agents (2-mercaptoethanol and bathocuproine sulfonate) to modified Iscove medium with 10% fetal bovine serum (HMI11) (11). HMI11 is now widely used for routine cell culture of laboratory strains of T. brucei and underpins many of the approaches employed in the search for new trypanocidal compounds. Nevertheless, HMI11 is a very rich medium compared to the natural in vivo environment of bloodstream form T. brucei, which may give...

Trypanosoma bruceimetabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion

“…The high consumption of tryptophan and phenylalanine by bloodstream form trypanosomes (19,21,22,25) suggested that the abnormal levels of aromatic ketoacids present in infected animals are transamination products generated by the parasites, but this has never been directly demonstrated. Therefore, aromatic ketoacid production by T. brucei incubated with tryptophan, tyrosine, or phenylalanine was measured directly using NAD-linked aromatic α-hydroxy acid dehydrogenase (26).…”

Metabolomic-Based Strategies for Anti-Parasite Drug Discovery

“…The new medium, CMM, provided comparable growth rates to HMI-11 and showed that parasites were much more sensitive to several drugs in a minimal medium where nutrients are not in excess. 17 These media are also useful for flux analyses in which stable-isotope labeled metabolites are tracked in real time through metabolic pathways. 4 In the absence of a minimal medium, defined media 21,22 are used to ensure that knowledge of the source of nutrients can be traced back to the cell under study or the medium in which the cells are grown.…”

“…[17][18][19][20] Metabolomics has recently been used to show that bloodstream-form T. brucei used only glucose, cysteine, glutamine, and aromatic amino acids from the HMI-11 classically used to culture bloodstream-form trypanosomes. 17 A minimal medium comprising these nutrients, salts, buffer, but still requiring serum was devised. The new medium, CMM, provided comparable growth rates to HMI-11 and showed that parasites were much more sensitive to several drugs in a minimal medium where nutrients are not in excess.…”

“…17 As a result, efforts have been made to create minimal media that are as close as possible to the most relevant biological environment. [17][18][19][20] Metabolomics has recently been used to show that bloodstream-form T. brucei used only glucose, cysteine, glutamine, and aromatic amino acids from the HMI-11 classically used to culture bloodstream-form trypanosomes. 17 A minimal medium comprising these nutrients, salts, buffer, but still requiring serum was devised.…”

“…Also, any metabolites that are in excess in the medium risk masking smaller changes to the internal metabolome if the medium is not removed. 17 Metabolite extraction generally coincides with the precipitation of membranes, proteins, and other macromolecular polymers from the cells. Metabolite extraction is usually achieved by the addition of a solvent such as methanol or acetonitrile in water for polar metabolites, often with chloroform added to improve the extraction of more hydrophobic metabolites.…”