Hmi 9

All transgenic T. bruceibrucei parasites used in this study were derived from monomorphic T. bruceibrucei 2T1 bloodstream forms (63 (link)) and were cultured in HMI-11 (HMI-9 [GIBCO] containing 10%, vol/vol, fetal bovine serum [GIBCO], Pen-Strep solution [penicillin at 20 U ml⁻¹, streptomycin at 20 mg ml⁻¹]) at 37°C and 5% CO₂ in vented flasks. Selective antibiotics were used: 5 μg ml⁻¹ blasticidin or hygromycin and 2.5 μg ml⁻¹ phleomycin or G418. RNAi was induced in vitro with tetracycline (Sigma-Aldrich) in 70% ethanol at 1 μg ml⁻¹. The endogenous Ty, mNeonGreen experiment was performed using the pPOTv6 vector (64 (link)). The generation of inducible TbCLK1 and KKT2 RNAi was done as previously described (20 (link)).

T. brucei bloodstream Lister 4,27 1339 cells were grown in HMI‐9 (Gibco, UK) medium supplemented with heat‐inactivated 15% FCS (Gibco, UK), at 37°C in a 5% CO₂ incubator. Cells were harvested during log phase (6 × 10⁵ – 1 × 10⁶ cells/ml). For growth curves, cells were counted every 24 h using an automated cell counter and split to 1 × 10⁵ cells/ml. Bloodstream form T. congolense cells (IL3000 strain) were grown and maintained at 37°C in a 5% CO₂ incubator in basal medium supplemented with complete Tc‐BSF3 medium. Basal medium consists of MEM supplemented with 50 mM acid HEPES, 52 mM NaHCO₃, 11 mM D‐Glucose, 2 mM sodium pyruvate, 0.08 mM adenosine, 0.2 mM hypoxanthine, 0.03 mM thymidine, and 0.05 mM bathocuproine sulfate acid. Basal medium pH was adjusted to 7.3 and filtered. To complete the medium, basal medium was supplemented with 15% FCS (Gibco, UK), 5% Serum Plus™ II Medium Supplement (Sigma, UK), 1.4% β‐mercaptoethanol, and 200 mM glutamine. Cells were harvested during log phase (5 × 10⁵ – 6 × 10⁶ cells/ml).

T. brucei cultures of the Lister 427 strain were maintained as both bloodstream (BSF) and procyclic (PCF) forms. BSF parasites were maintained at 37°C, 5% CO₂ in HMI-9 (GIBCO) medium supplemented with 20% fetal bovine serum (GIBCO). Cell passages were performed every 48 hours, with population density maintained between 1 x 10⁵ and 2 x 10⁶ cells.mL^-1. PCF cells were maintained at 27°C in SDM-79 (GIBCO) medium supplemented with 10% fetal bovine serum (GIBCO). Weekly passages were performed, but cell density was never lower than than 5 x 10⁵ cells.mL^-1.
Epimastigote forms of the CL Brener clone of T. cruzi were maintained in logarithmic growth phase at 28°C in liver infusion tryptose (LIT) medium supplemented with 10% fetal bovine serum (GIBCO) and penicillin (10,000 U.mL^-1)/Streptomycin (10,000 μg.mL^-1) (GIBCO) as described by [41 (link)]. Metacyclic trypomastigotes, obtained after metacyclogenesis of epimastigotes cultures maintained in LIT medium for 15 to 20 days, were used to infect Vero cells cultured in DMEM medium (GIBCO) supplemented with 5% fetal bovine serum (GIBCO).

Monomorphic Trypanosoma brucei brucei 2T1 bloodstream forms^{49 (link)} were cultured in HMI-11 [HMI-9 (GIBCO) + 10% v/v foetal bovine serum (GIBCO), Pen/Strep solution (penicillin 20 U ml⁻¹, streptomycin 20 mg ml⁻¹)] at 37 °C/5% CO₂ in vented flasks. Selective antibiotics were used as follows: 0.2 μg ml⁻¹ puromycin, 0.5 μg ml⁻¹ phleomycin (InvivoGen), 2.5 μg ml⁻¹ hygromycin B (Calbiochem). RNAi was induced in vitro with tetracycline (Sigma Aldrich) in 70% ethanol at 1 μg ml⁻¹.
To test for serum resistance 200 μl of log-phase parasites in HMI11 at 10⁶ cells ml⁻¹ were exposed to fresh rat serum for 3 h (10% and 50% concentration). Relative survival was measured of induced (for 48 h) and uninduced clones compared to no-serum controls. The whole experiment was performed in triplicate.
To test for response to osmotic shock^{18 (link)} 2 × 10⁶ cells were resuspended for 5 min at 4 °C in 0.5 ml of a solution 55 mM KCl/1 mM glucose to induce cell swelling. 0.5 ml of a 263 mM KCl/1.75 mM Mg₂Cl shrinking solution was added on top and cells incubated for 5 min at 4 °C. Remaining cells were washed once and resuspended in 1 ml of HMI11 media before counting. Survival of the induced cells compared to uninduced controls was plotted and significant results (p < 0.05) after T-tests indicated with a star.

The parasite used for this study was the bloodstream form trypomastigotes of Trypanosoma brucei subsp. brucei, Strain Lister 427 VSG 221 kindly donated by BEI resources (https://www.beiresources.org/ accessed on 4 April 2019). Parasites were axenically cultivated in sterile vented flasks containing complete Hirumi’s modified Iscove’s medium 9 (HMI-9) [500 mL IMDM (Iscove’s modified Dulbecco’s medium) (Gibco, Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (HIFBS) (Sigma Aldrich), 10% (v/v) serum plus (Sigma Aldrich), HMI-9 supplement (1 mM hypoxanthine, 0.16 mM thymidine, 50 µM bathocuproine disulfonic acid, 1.5 mM cysteine, 1.25 Mm pyruvic acid, 0.2 mM 2-mercaptoethanol (Sigma Aldrich)), and 1% (v/v) penicillin–streptomycin (Sigma Aldrich) and incubated at 37 °C in a 5% CO₂ atmosphere. Cultures were routinely monitored every 72 h using a Lumascope LS520 inverted fluorescence microscope (Etaluma, Inc., USA) to assess parasite density and subsequently passaged with fresh complete medium in such a way that the cell density never exceeded 2 × 10⁶ cells.mL⁻¹ [58 (link)].

T. brucei cultures of the Lister 427 strain bloodstream (BSF) form were maintained at 37°C, 5% CO₂ in HMI-9 (GIBCO) medium supplemented with 20% fetal bovine serum (GIBCO). Cell passages were performed every 48 h, with population density maintained between 1 × 10⁵ and 2 × 10⁶ cells/mL. Epimastigotes of the CL Brener clone of T. cruzi were maintained in logarithmic growth phase at 28°C in liver infusion tryptose (LIT) medium supplemented with 10% fetal bovine serum (GIBCO) and penicillin (10,000 U/mL)/Streptomycin (10,000 μg/mL) (GIBCO) as described (Camargo, 1964 (link)). Metacyclic trypomastigotes, obtained after metacyclogenesis of epimastigote cultures maintained in LIT medium for 15–20 days, were used to infect Vero cells cultured in high-glucose DMEM medium (GIBCO) supplemented with 5% fetal bovine serum (GIBCO) and penicillin (10,000 U/mL)/Streptomycin (10,000 μg/mL) (GIBCO).