Biopterin

Two isogenic clones of L. guyanensis (MHOM/BR/75/M4147) showing uniformly high levels of LRV1 or completely lacking it were described previously 55 (link). We refer to these clones as LgyLRV1+ and LgyLRV1-, respectively. Both strains express a firefly luciferase gene (LUC), integrated stably into the locus of the small ribosomal subunit and displayed comparable luminescent efficacy 55 (link). In addition, two strains of L. infantum were used. The first one was isolated from a dog in Spain (JPC MCAN/ES/98/LLM-722) and will be referred as Linf S1, while the second one was isolated from a human patient in Switzerland (MHOM/CH/2016/BELA) and will be referred as Linf S2. Parasites were cultured in vitro as promastigotes at 26°C in freshly prepared Schneider’s insect medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (PAA®), 10mM HEPES and 50U/ml penicillin/streptomycin (Animed®), 0.6mg/L biopterin and 5mg/L hemin (Sigma-Aldrich®). Each passage yielded infectious metacyclic promastigotes after 6 days and stocks were kept no longer than 5 passages.

The two isogenic clones of L.g infected with, or depleted of LRV1 (termed LRV1+ or LRV1- respectively), used in this study were described and characterized previously [65 (link)]. Briefly, these lines were derived from the LRV1+ parent strain, L.g M4147 (MHOM/BR/75/M4147) or an LRV1-null derivative [66 (link)]. The parasites express similar levels of a firefly luciferase (5x10⁷ photons/sec/10⁶ parasites) from a LUC gene integrated stably into the small subunit gene of the ribosomal RNA locus.
Parasites were cultured in vitro as promastigotes at 26°C in freshly prepared Schneider’s insect medium (Sigma) supplemented with 10% heat-inactivated foetal bovine serum (PAA), 10mM HEPES and 50U/ml penicillin/streptomycin (Animed), 0.6 mg/L biopterin and 5 mg/L hemin (Sigma-Aldrich). Each passage yielded infectious metacyclic promastigotes after 6 days and stocks were kept for no longer than 5 passages.

Leishmania mexicana (isolate MNYC/BZ/62/M379) promastigotes were grown in M199 medium supplemented with 2 μg/ml biopterin, 2 μg/ml hemin (all from Sigma-Aldrich, St. Louis, USA), 25 mM HEPES (Lonza, Basel, Switzerland), 50 units/ml of Penicillin/Streptomycin (Life Technologies/Thermo Fisher Scientific, Carlsbad, USA) and 10% heat-inactivated fetal bovine serum (BioSera Europe, Nuaillé, France) at 23°C.
Throughout the long-term experiment, WT and ΔKu80 cell cultures were passaged 2 times a week for a total of 100 passages. Cells were sampled from passage numbers 0, 25, 50, 75, and 100.
Growth kinetics in vitro was analyzed as described previously [36 (link)]. The statistical significance was evaluated using unpaired t test in Prism v. 8.0.1 (GraphPad Software, San Diego, USA).

Previously we described a clonal derivative of the LRV1+ strain of L. guyanensis M4147 (MHOM/BR/75/M4147), containing a firefly luciferase (ffLUC) gene integrated stably into the small subunit gene of the ribosomal RNA locus (LgM4147/SSU:IR2SAT-LUCb LRV1+) [27 (link)]. In work to be described elsewhere, clonal lines retaining or lacking LRV1 were obtained following brief drug treatments [28 (link)], resulting in isogenic LRV1+ or LRV1- lines. These lines fully recapitulate the LRV1+ versus LRV1- phenotypes found in prior studies [7 (link)] using isogenic lines developed by Ro and Patterson after transfection, drug treatment and prolonged culture in vitro (Kuhlman and Beverley, in preparation). Parasites were cultured in vitro as promastigotes at 26°C in freshly prepared Schneider’s insect medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (PAA), 10mM HEPES and 50U/mL penicillin/streptomycin (Animed), 0.6mg/L biopterin and 5mg/L hemin (Sigma-Aldrich). Each passage yielded infectious metacyclic promastigotes after 6 days and cultures were kept no longer than 5–6 passages.

Standard solutions for acylcarnitine and amino acid profiling, methanol, formic acid, bovine serum albumin (BSA), sodium chloride, 6-hydroxynicotinic acid, 3-indole acrylic acid, neopterin, biopterin, l-tryptophan, and ascorbic acid were received from Sigma–Aldrich (USA). Acetonitrile was obtained from Chromasolv^® (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). Ultra-pure water was obtained from a Millipore Milli-Q water purification system (Millipore Corporation, Billerica, MA, USA). Isotope-labeled standard solutions of metabolites related to tryptophan catabolism were received from Toronto Research Chemicals (Toronto, ON, Canada). Isotope-labeled standard solutions for acylcarnitine and amino acid profiling were purchased from MassChrom Amino Acids and Acylcarnitines Non-Derivatised 57,000 Kit (Chromsystems, Germany).

L. major (Lv39c5) promastigotes were grown at 26 °C in M199 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 25 mM HEPES (pH 7.2; Sigma-Aldrich), 0.1 mM adenine (Sigma-Aldrich), 0.0005% (w/v) hemin (Sigma-Aldrich), 2 mg/mL biopterin (Sigma-Aldrich), 0.0001% (w/v) biotin (Sigma-Aldrich), 10% (v/v) heat-inactivated fetal bovine serum (Gibco Laboratories, Grand Island, NY, USA), and an antibiotic cocktail (50 U/mL penicillin, 50 mg/mL streptomycin) (Sigma-Aldrich). L. major cultures used for qPCR analysis were grown in Schneider’s medium (Gibco Laboratories) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco Laboratories) and 40 μg/mL gentamicin (Sigma) at 26 °C.