Gentamicin

A recombinant strain of L. tarentolae parasites expressing A2 and CP genes was already available from our previous study (30 (link)). The L. tarentolae and recombinant L. tarentolae A2-CPA-CPB^-CTE parasites were grown in M199 (Sigma, Missouri, USA) supplemented with 5% heat-inactivated fetal calf serum (FCS, Gibco) and 0.1 mM adenosine, 40 mM HEPES (pH 7.2), 5 μg/mL hemin (all chemicals purchased from Sigma), and 50 μg/mL gentamicin (Biosera, France) at 26 °C. The L. infantum strain MCAN/ES/98/LLM-877 was kindly provided by WHO collaborating center for leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain and kept in virulent state by continuous passage in hamsters. Amastigotes were isolated from the previously-infected hamsters’ spleens and cultured in Novy-MacNeal-Nicolle (NNN) media and prepared for the infectious challenge as described previously (30 (link)). Parasites were frozen and thawed (F/T) and protein concentration of the lysate was determined by bicinchoninic acid reagent (BCA, PIERCE, Chemical Co. Rochford III).

HT-29, HCT 116, SW620, CCD-18Co and CCD-1072Sk cell cultures were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). HT-29 and SW620 cells were cultured in complete RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA), HCT 116 cells in McCoy’s medium (PAN-Biotech GmbH, Aidenbach, Germany), CCD-18Co and CCD-1072Sk in MEM medium (Biosera, Nuaille, France). All cultivation media were supplemented with 10% fetal bovine serum (Biosera, Nuaille, France) and antibiotics (1% antibiotic-antimycotic 100 $\times$ and 50 μg/mL gentamicin; Biosera, Nuaille, France).

HT-29, HCT 116, and CT26.WT cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). HT-29 and CT26.WT cells were cultured in complete RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) and HCT 116 cells in complete McCoy’s medium (PAN-Biotech GmbH, Aidenbach, Germany). Both cultivation media were supplemented with 10% fetal bovine serum (FBS; Biosera, Nuaille, France) and antibiotics (1% antibiotic-antimycotic 100× and 50 µg ml⁻¹ gentamicin; Biosera, Nuaille, France) at 37 °C, 95% humidity, and in an atmosphere of 5% CO₂.

Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin and gentamicin were all obtained from Biosera (Nuaillé, France). Type II collagen purchased from Sigma Aldrich (St. Louis, MO, USA). All other chemicals used were of the best commercially available grade.

TR146 cell line was purchased from Public Health England, UK. Human recombinant insulin, L-arginine (non-animal source, cell culture tested), L-lysine (≥97%), L-histidine (≥Reagent Plus 99%), L-glutamic acid (minimum 99% TLC), L-aspartic acid (99%), sodium deoxycholate, thiazolyl blue tetrazolium bromide (methylthiazolydiphenyl-tetrazolium bromide; ≥97%; enzymatic), dimethyl sulfoxide (DMSO) and trifluoroacetic acid were purchased from Sigma-Aldrich, UK. Hanks balanced salt solution (HBSS), Fetal bovine saline (FBS) were obtained from Gibco Lab., UK. Acetoniltrile and absolute ethanol were purchased from Fisher scientific, UK. Hank’s balanced salt solution (HBSS), fetal bovine serum (FBS), Hams F-12 nutrient mix and trypsin-EDTA were obtained from Gibco^® Lab., UK. Gentamicin and penicillin/ streptomycin were obtained from Bio Sera, UK. Acetoniltrile and absolute ethanol were purchased from Fisher scientific, UK. 6 well plates, 6 well polycarbonate transwell inserts and 12 well polyester transwell inserts were purchased from Appletonwoods, UK. All the chemicals and reagents were used as obtained. All water used was double-distilled and autoclaved.

The fragmented biopsy from the border of the ulcers were collected and inoculated into Schneider’s Drosophila medium (Sigma, Darmstadt, Germany), supplemented with 10% heat- inactivated fetal bovine serum (FBS, Gibco, UK), 40 mM HEPES, 2 mM, 0.1 mM adenosine, 2 mM L-glutamine, 0.5μg/ml hemin (all from Sigma, Germany) and 50 μg/ml gentamicin (Biosera, France) and incubated at 26°C. Cultures were checked microscopically for live and motile promastigotes for maximum of 3–4 weeks.