A Luciferase-Expressing Leishmania braziliensis Line That Leads to Sustained Skin Lesions in BALB/c Mice and Allows Monitoring of Miltefosine Treatment Outcome

Abstract

Background: Leishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine.

Methodology/principal findings: A L. braziliensis line, originally isolated from a cutaneous leishmaniasis patient, was passaged repeatedly in laboratory rodents and further genetically manipulated to express luciferase. Once collected from a culture of parasites freshly transformed from amastigotes, 106 wild type or luciferase-expressing stationary phase promastigotes were inoculated subcutaneously in young BALB/c mice or golden hamsters. In both groups, sustained cutaneous lesions developed at the site of inoculation, no spontaneous self- healing being observed 4 months post-inoculation, if left untreated. Compared to the wild type line features, no difference was noted for the luciferase-transgenic line. Infected animals were treated with 5 or 15 mg/kg/day miltefosine orally for 15 days. At the end of treatment, lesions had regressed and parasites were not detected. However, relapses were observed in animals treated with both doses of miltefosine.

Conclusions/significance: Here we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing L. braziliensis line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration.

Fig 1. Characterisation of a transgenic line of L. braziliensis expressing luciferase.

(A) Schematic representation of the linear cassette for integration in the SSU rDNA locus. SSU, small subunit; SAS, splice acceptor site; luc2, luc2 coding sequence; α-tub IR, intergenic region of the L. enrietii α-tubulin gene; hyg, hygromycin phosphotransferase gene; CPB IR, L. mexicana cysteine protease B gene intergenic region. Position of the primers and size of amplified fragments are indicated by the dotted lines. (B) Size separation of PCR products amplified using primers complementary to the luc2 ORF extremities from genomic DNA purified from four independent transfected clones (lanes 1 to 4) and from the wild type strain (lane 5). (C) Agarose gel electrophoresis of amplified products obtained from Lb-WT (lane 1) or Lb-LUC (lane 2) with the pairs of primers S1/luc2R or luc2i/S4. (-) indicates the control reaction without template DNA. (D) Luminescence intensity and number of Lb-LUC promastigotes. Promastigotes were serially diluted and luminescence was measured using a microplate reader, after addition of luciferin. RLU, relative light units.

Fig 2. Course of disease after L. braziliensis H3227 inoculation in hamsters and BALB/c mice.

Evolution of lesion size in hamsters (A) and BALB/c mice (B) inoculated with Lb-LUC or Lb-WT H3227 strain. Animals were inoculated with 10⁶ stationary-phase promastigotes in the left hind footpad and lesion size was measured weekly (five animals per group). Data is the mean and standard deviation from one experiment representative of two independent experiments. Student’s t test comparing the area under the curve detected non-statistically significant differences with P = 0.25 (A) and P = 0.11 (B). Representative images of a hamster at the 5^th week (C) and of a BALB/c mouse at the 6^th week post-inoculation (D) with H3227 Lb-LUC parasites. The left footpad (inoculated) and right footpad (control) of a hamster (C) and a BALB/c mouse (D), representative of each group of animals, are shown.

Fig 3. In vivo efficacy of miltefosine in a L. braziliensis-inoculated BALB/c mice model.

10⁶ Lb-LUC stationary promastigotes were injected in the left hind footpad and animals were treated with 5 or 15 mg/kg/day of miltefosine for 15 consecutive days (from to 4^th to the 6^th week post-inoculation). Average lesion size was measured weekly (five animals per group) in untreated and treated animals and horizontal black bar indicates MF treatment (A). Parasite burden was evaluated by bioluminescence at the end of the treatment (6^th week post-inoculation) (B), at the 17^th (C) and at the 23^th week post-inoculation (D) (40, 120 and 160 days post-inoculation respectively). Statistical analysis was performed with One Way ANOVA, followed by the Tukey post-test. * p < 0.05; **, p < 0.001. Ph/sec/cm²/sr, photons per second per square centimetre per steradian.

Fig 4. Parasite burden of left hind footpad of BALB/c mice treated with MF and evaluated by bioluminescence.

Groups of 5 animals untreated (NT), or treated with 5 or 15 mg/kg/day MF (MF 5 and MF 15 respectively) were evaluated at 40, 120 and 160 days (6^th, 17^th and 23^th week respectively) post-inoculation (pi). The bars on the right show a pseudo-colour scale representing light intensities.