Appraisal of a Leishmania major Strain Stably Expressing mCherry Fluorescent Protein for Both In Vitro and In Vivo Studies of Potential Drugs and Vaccine against Cutaneous Leishmaniasis
Background Leishmania major cutaneous leishmaniasis is an infectious zoonotic disease. It is produced by a digenetic parasite, which resides in the phagolysosomal compartment of different mammalian macrophage populations. There is an urgent need to develop new therapies (drugs) against this neglected disease that hits developing countries. The main goal of this work is to establish an easier and cheaper tool of choice for real-time monitoring of the establishment and progression of this pathology either in BALB/c mice or in vitro assays. To validate this new technique we vaccinated mice with an attenuated Δhsp70-II strain of Leishmania to assess protection against this disease. ; Methodology We engineered a transgenic L. major strain expressing the mCherry red-fluorescent protein for real-time monitoring of the parasitic load. This is achieved via measurement of fluorescence emission, allowing a weekly record of the footpads over eight weeks after the inoculation of BALB/c mice. ; Results In vitro results show a linear correlation between the number of parasites and fluorescence emission over a range of four logs. The minimum number of parasites (amastigote isolated from lesion) detected by their fluorescent phenotype was 10,000. The effect of antileishmanial drugs against mCherry+L. major infecting peritoneal macrophages were evaluated by direct assay of fluorescence emission, with IC50 values of 0.12, 0.56 and 9.20 µM for amphotericin B, miltefosine and paromomycin, respectively. An experimental vaccination trial based on the protection conferred by an attenuated Δhsp70-II mutant of Leishmania was used to validate the suitability of this technique in vivo. ; Conclusions A Leishmania major strain expressing mCherry red-fluorescent protein enables the monitoring of parasitic load via measurement of fluorescence emission. This approach allows a simpler, faster, non-invasive and cost-effective technique to assess the clinical progression of the infection after drug or vaccine therapy. ; This research was supported by Ministerio de Ciencia y Tecnología (grants AGL2010 16078/GAN), Instituto de Salud Carlos III (grant PI09/0448 and the Network of Tropical Diseases RICET RD06/0021/1004). RAV, CFP and ECA are pre-doctoral fellows granted by RICET (ISCIII), Junta de Castilla y León (ESF; European Social Founding) and University of León, respectively to RMR. Instituto de Salud Carlos III (Network of Tropical Disasese RICET RD06/0021/0016), Ministerio de Ciencia e Innovación (SAF2010-17833), ChagasEpiNet 223034 European Union Seventh Framework Programme and Fundación Ramón Areces to MF. Funding by ISCIII-RETIC RD06/0021/0008-FEDER to JMR and ISCIII-RETIC RD06/0021/0006-FEDER to LR is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ; Peer reviewed
