INTRODUCTION
Leishmaniasis is a worldwide-expanded zoonotic disease caused by the infection with protozoan parasites of the genus Leishmania (Ross, 1903), which include 30 species classified in two subgenera, Leishmania and Viannia; approximately 20 are pathogenic for humans (WHO, 2010). These species generally present different epidemiological and clinical characteristics related to different genetic and phenotypic profiles. The clinical manifestation of the disease depends on the specie that is infecting. In the new world seven species of the parasite have been associated with localized, disseminated or diffuse cutaneous but also mucocutaneous leishmaniasis: Leishmania braziliensis, Leishmania panamensis, Leishmania guyanensis and Leishmania peruviana, belong to the subgenus Viannia and Leishmania mexicana and Leishmania amazonensis belonging to Leishmania subgenus (WHO, 2010).
Current chemotherapy agents for leishmaniasis include pentavalent antimonials, miltefosine, pentamidine and amphotericin B (WHO, 2010). However, these drugs are either toxic, expensive, or both, interfering with the effective treatment of people around the world and therefore more and better antileishmanial drugs are currently needed. The identification of new potential drug compounds and molecular targets throughout in vitro and in vivo systems are the first steps toward new therapeutic approaches and rational drug design strategies. Development of new therapeutic approaches for leishmaniasis treatment requires not only accurate and precise methodologies, but also well-known biological system to measure cytotoxicity and antileishmanial activity of the new compounds both in vitro and in vivo.
During the last decade, more accurate and precise methodologies have been developed and implemented through the use of recombinant parasites expressing diverse reporter genes such as β-lactamase, β-galactosidase, firefly luciferase or green fluorescent protein (GFP) genes. This recombinant parasites are used for in vitro high-through put methods and in vivo assays for antileishmanial drug screening (Buckner et al. Reference Buckner, Verlinde, La Flamme and Van Voorhis1996; Roy et al. Reference Roy, Dumasm, Serenom, Wum, Singhm, Tremblay, Ouellette, Olivier and Papadopoulou2000; Buckner and Wilson, Reference Buckner and Wilson2005; Lang et al. Reference Lang, Goyard, Lebastard and Milon2005; Bolhassani et al. Reference Bolhassani, Taheri, Taslimi, Zamanilui, Zahedifard, Seyed, Torkashvand, Vaziri and Rafati2011; Pulido et al. Reference Pulido, Munoz, Restrepo, Mesa, Alzate, Vélez and Robledo2012). Although in spite that it is known that the infectivity and susceptibility of the different Leishmania species to the established and experimental drugs is variable even in the same Leishmania species, reports come from several studies, each of them done by different authors and using different methodologies, parasite biological stages or Leishmania species and strains, making difficult the generation of adequate conclusions.
Here, the infectivity and sensitivity to commonly used antileishmanial drugs was tested in transfected L. braziliensis, L. panamensis, L. guyanensis, L. mexicana and L. amazonensis, the most prevalent Leishmania species causing cutaneous leishmaniasis in Colombia and Latin America, expressing the GFP gene (EGFP). Differences and similarities related to the growth characteristics in culture as promastigotes, the infectivity and the sensitivity to current antileishmanial drugs of these Leishmania species are discussed.
MATERIALS AND METHODS
Cell line and culture conditions
U-937 promonocytes (CRL1593.2™) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in standar conditions at 37 °C, 5% CO2, with change of medium every 3 days until use. U-937 cells (suspension) were cultured in RPMI-1640 with 10% fetal bovine serum (FBS) and 1% antibiotics (100 U mL−1 penicillin and 0·1 mg mL−1 streptomycin).
Leishmania strains and in vitro culture
The EGFP-transfected Leishmania species were made in a previous work (see Pulido et al. Reference Pulido, Munoz, Restrepo, Mesa, Alzate, Vélez and Robledo2012). During selection of fluorescent parasites, promastigotes were grown in Schneider's medium supplemented with 10% FBS, 1% antibiotics and nourseothricin (50 or 100 µg mL−1 to Viannia or Leishmania subgenus, respectively). Then, both wild-type and EGFP transfectants L. panamensis (MHOM/CO/87/UA140), L. braziliensis (MHOM/CO/88/UA301), L. guyanensis (MHOM/CO/CL007), L. mexicana (MHOM/MX/95/NAN1) and L. amazonensis (IFLA/BR/67/PH8) were grown at 26 °C in biphasic medium of Novy–MacNeal–Nicholle (NNN) medium and an overlay of phosphate buffered saline-phosphate-buffered saline (PBS) and glucose, pH 6.9.
Growth curves
Three days old promastigotes were harvested and adjusted at 5 × 104 parasites mL−1 liquid phase. One mL were placed into each well of 24 well-plate containing the NNN medium. Parasites were incubated at 26 °C during 12 days. Daily, liquid phase of two wells was harvested and diluted 1:10 in PBS, parasites were counted in an haemocytometer and the parasite average determined for each day to build growth curve.
Evaluation of macrophage infection
To establish the maximal amounts of parasites : cell ratio that gives the 50% infection of cells, commonly named Infectious Dose 50 (ID50), U-937 cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA), pH 7.2 supplemented with 10% FBS and 1% antibiotics at 37 °C, 5% CO2. After 2–3 days growth the U937 cells were washed with a PBS solution and adjusted at 3 × 105 cells mL−1 of RPMI medium containing 0·1 µg mL−1 of phorbolmyristateacetate (PMA) (Sigma-Aldrich, St Louis, MO, USA). The cells were dispensed in 24-well cell-culture plate with and without a sterile 12 mm diameter cover slide glass, then plates were incubated 72 h at 37 °C and 5% CO2. Subsequently the cells were infected with early stationary phase promastigotes at 5 : 1, 10 : 1, 20 : 1, or 40 : 1 parasite : cell ratio following a previously described methodology (Robledo et al. Reference Robledo, Valencia and Saravia1999). After incubation for 3 h at 34 °C, extracellular promastigotes were removed by four washes with pre-warmed fresh medium and plates were incubated during 24 additional hours. Then, medium was removed and the cells were washed twice with 1 mL cold PBS, cells were fixed with methanol and stained with 10% Giemsa (Merck S. A, Bogotá, Colombia) and analysed under a light microscope (1.000×) to determine the infection percentage. Each dose of parasites was tested in triplicate in at least two independent experiments. Infectivity of each Leishmania species was determined according to the infected cells percentages obtained for each dose of parasites. The results were expressed as the ID50 calculated by the Probit method (Finney, Reference Finney1978).
In vitro cytotoxicity of antileishmanial drugs on U937 cells
The cytotoxic activity of antileishmanial drugs meglumine antimoniate, miltefosine, pentamidine isethionathe and amphotericin B was assessed based on the cell growth (viability) of U937 cells and evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method as described previously (Taylor et al. Reference Taylor, Muñoz, Cedeño, Vélez, Jones and Robledo2010). Briefly, into each well of a 96-well cell-culture dishes were dispensed 100 000 cells 100 µL−1 in RPMI-1640 supplemented with 10% FBS and 100 µL of the corresponding concentrations of antileishmanial drugs. Six double serial diluted concentrations were evaluated starting at 200 µg mL−1. The cells were incubated at 37 °C with 5% CO2 for 72 h in the presence of each antileishmanial drug, and then the effect was determined by measuring the activity of the mitochondrial dehydrogenase by adding 10 µL well−1 of MTT solution (0·5 mg mL−1) and incubating at 37 °C for 3 h. The reaction was stopped by adding 100 µL well−1 of dimethylsulfoxide for 30 min. Cell growth was determined based on the quantity of formazan produced, which was measured at 570 nm in a reader plate spectrophotometer (Varioskan Flash, Thermo Scientific, Waltham, MA, USA). Cells cultured in the absence of any antileishmanial drug were used as cell growth controls (negative control), while cells cultured in presence of doxorubicin were used as cytotoxicity control (positive control). Each concentration was tested in triplicate in at least two independent experiments.
Cytotoxicity was determined according to the percentages of cell growth (viability) obtained for each tested compound or medium alone. Percentages of viability were calculated using equation (1):
$$\eqalign{\% \;{\rm Viability} = & [({\rm O.D. \; of \; treated \; cells})/ \cr & (\rm O.D. \; of \; untreated \; cells)] \times 100,} $$
where the O.D. of the untreated cells corresponds to 100% viability. In turn, the percentage of cell growth inhibition is calculated using the equation (2):
$${\rm \% }\;{\rm Cell}\;{\rm growth}\;{\rm inhibition = 100}{- }{\rm \% }\;{\rm of}\;{\rm Viability}{\rm .}$$
Percentage of cell growth inhibition was used to calculate the lethal concentration 50 (LC50) that corresponds to the concentration of drug that gives the half-maximal inhibition of the cell growth by the Probit method (Finney, Reference Finney1978). Cytotoxicity of antileishmanial drugs was graded according to the LC50 values, as follows: High cytotoxicity: LC50 < 50 µg mL−1; Moderate cytotoxicity LC50 > 50 but <200 µg mL−1 and potential non-cytotoxicity: LC50 > 200 µg mL−1.
In vitro leishmanicidal activity on intracellular amastigotes
The effect of antileishmanial drugs against intracellular amastigotes of Leishmania species was evaluated by flow cytometry using the methodology described by others (Taylor et al. Reference Taylor, Muñoz, Cedeño, Vélez, Jones and Robledo2010; Pulido et al. Reference Pulido, Munoz, Restrepo, Mesa, Alzate, Vélez and Robledo2012). Intracellular amastigotes were obtained after infection of U937 cells with promastigotes of each Leishmania species; in brief, U937 cells were dispensed in 24-well plates at a concentration of 300 000 cells per well and were treated with 1 µM of PMA for 72 h at 37 °C. Then, cells were infected with promastigotes in stationary growth phase (day 5) at a ratio previously established for each Leishmania species and incubated 3 h at 34 °C, 5% CO2. Cells were washed twice with PBS to eliminate non-internalized parasites and fresh RPMI-1640 was added into each well (1 mL); plates were incubated again at 34 °C and 5% CO2 to allow intracellular differentiation to amastigotes form. After 24 h of infection of U937 cells, culture medium was replaced by fresh RPMI-1640 medium containing meglumine antimoniate, miltefosine, pentamidine or amphotericin B at the corresponding concentration (4-fold dilutions that is prepared starting at a concentration not exceeding the LC50, as previously determined). Infected and treated cells were maintained at 34 °C and 5% CO2 for 72 h. After 72 h of incubation at 37 °C, 5% CO2 cells were removed using trypsin/EDTA solution and washed twice with PBS by centrifuging 10 min at 1100 rpm, 4 °C. Then, cells were analysed in an Argon laser flow cytometer (Cytomics FC 500MPL, Beckman Coulter. Pasadena, CA, USA) by reading at 488 nm excitation and 525 nm emission. Ten thousand events were counted from each well. The percentage of infected cells was determined by dot plot analysis and the mean fluorescence intensity in those infected cells by using histogram analysis (Pulido et al. Reference Pulido, Munoz, Restrepo, Mesa, Alzate, Vélez and Robledo2012). Infected cells incubated in culture RPMI 1640 medium alone were used as control for infection. Each concentration was assessed in triplicate in at least two independent experiments.
Antileishmanial activity was determined according to the reduction of infected cells percentages obtained for each experimental condition. The infection percentage and infection inhibition for each concentration of each compound were calculated according to equation (3):
$$\eqalign{ \% \;&{\rm Infection} = \% \;{\rm Infected}({\rm treated}\;{\rm cells})/\% \cr & {\rm Infected}\;({\rm untreated}\;{\rm cells}) \times 100.} $$
In turn, the percentage of inhibition was calculated using equation (4):
$${\%}\;{\rm Inhibition} = 100 - \% \;{\rm of \; infection}.$$
The results were expressed as the effective concentration 50 (EC50) that corresponds to the concentration of drug that gives the half-maximal inhibition of the intracellular parasites calculated by the Probit method from % of inhibition data (Finney, Reference Finney1978). The degree of antileishmanial activity was established as convenience according to the EC50 values, as follows: High activity: EC50 < 20 µg mL−1; moderate activity: EC50 > 20 but <70 µg mL−1; and low activity: EC50 > 70 µg mL−1. The index of selectivity (IS) was calculated by dividing the cytotoxicity and the antileishmanial activity using the equation (5):
$${\rm IS} = {\rm L}{\rm C}_{{50}}{\; (\rm Cytoxicity)/{\rm }E}{\rm C}_{{50}}{ \; (\rm Activity)}.$$
Statistical analysis
Parasite growth curves were performed in duplicate, while the in vitro experiments for ID50, LC50 and EC50 were performed in triplicate in at least two independent assays. Data represent the mean value ± s.d. The statistical significance of differences between experimental groups was determined using Mann–Whitney, Wilcoxon matched pair or Student's t-test were used when suitableusing Graph Pad Prism 6 software (San Diego CA, USA). A P-value below 0·05 was considered statistically significant.
RESULTS
Leishmania species growth curve
Results were obtained by counting daily the promastigotes number of each Leishmania strain (wild-type and EGFP transfectant) grown in NNN medium. Standar deviations for each test point were below 5% of the corresponding mean values. Growth curves for promastigotes of both wild-type and EGFP-Leishamnia strains are showed in Fig. 1. With the initial population densities of 5·0 × 104 parasites mL−1 all the evaluated species of Leishmania entered into the stationary phase at the same time. Nevertheless, the number of parasites at the stationary phase was always lower with L. panamensis-EGFP and L. braziliensis-EGFP than L. mexicana-EGFP, L. amazonensis-EGFP and L. guyanensis-EGFP. Growth curves of L. amazonensis-EGFP and L. mexicana-EGFP were very similar among them.
Growth of Leishmania species in NNN biphasic medium. Data represent X + s.d. of parasites amount per day of growth. NNN, Novy–MacNeal–Nicholle
Differences in the growth rate were observed between L. panamensis-EGFP and L. braziliensis-EGFP in comparison with L. guyanensis-EGFP, L. amazonensis-EGFP and L. mexicana-EGFP. After 2 or 3 days, Leishmania promastigotes growth of all species was exponential and peak of growth was reached after 5 days; on day 6 promastigotes motility and refraction of L. panamensis started to decrease until day 9 (Fig. 1). In L. amazonensis-EGFP, motility of parasite started to decrease until day 8. In turn, early exponential growth of L. guyanensis-EGFP and L. mexicana-EGFP, achieved in day 3 with stationary phase occurring in days 5–6 or 5–7 of growth, respectively, and decrease of motility and refraction occurred in days 9 and 11, respectively. Lastly, in L. braziliensis-EGFP the stationary phase was achieved in days 5–7 and growth decreased occurred in day 11.
Infectivity of Leishmania species
Leishmania braziliensis-EGFP showed higher infective capability evidenced by lower amounts of promastigotes needed to achieve the 50% of infection of the cell population (Table 1). The infective capability of L. mexicana-EGFP and L. amazonensis-EGFP was slightly lower than L. braziliensis-EGFP. In turn, L. panamensis-EGFP and L. guyanensis-EGFP showed the lowest infective capability because these Leishmania species requires higher amount of parasites to infect 50% of cells. The infective capability of L. panamensis-EGFP and L. guyanensis-EGFP was statistically different than that showed by L. braziliensis-EGFP, L. mexicana-EGFP and L. amazonensis-EGFP.
Infectivity of Leishmania species to human U-937 macrophages
Data represent X + s.d. of the Infective Dose 50 (ID50) in Leishmania expressing the GFP gene (EGFP) and wild-type (WT) of at least two independent experiments.
Sensitivity of human U937 macrophages to antileishmanial drugs
The sensitivity of human U937 cells to meglumine antimoniate, miltefosine, pentamidine isethionate and amphotericin B was assessed to identify the maximum concentration of drug to which cells should be exposed. The U937 macrophages were highly sensitive to pentamidine isethionate (LC50 8·3 ± 1·1 µg mL−1) and moderately sensitivity to miltefosine and amphotericin B (LC50 104·6 ± 11·7 and 137·7 ± 0·6 µg mL−1, respectively). The U937 cells were not susceptible to meglumine antimoniate, showing a LC50 value of >200·0 µg mL−1.
Sensitivity of Leishmania species to current antileishmanial drugs
The sensitivity of Leishmania parasites to the antileishmanial drugs currently used to treat leishmaniasis varied among Leishmania species and type of drug. As shown in Table 2, all Leishmania strains were highly sensitive to amphotericin B with small differences in the EC50 values that were not statistically significant(P > 0·01). On the contrary, all EGFP-transfected Leishmania species showed a moderate sensitivity to miltefosine with EC50 values ranging from 36·3 ± 1·1 to 88·7 ± 3·1 µg mL−1, L. panamensis-EGFP was the most sensitive to pentamidine isethionate followed by L. amazonensis-EGFP and L. braziliensis-EGFP (EC50 values <28·0 µg mL−1), while L. guyanensis-EGFP and L. mexicana-EGFP were less sensitive (EC50 > 40·0 µg mL−1). Differences in sensitivity to pentamidine isethionate were statistically significant (P < 0·01). Overall, amphotericin B was the most active antileishmanial drug for all EGFP-transfected Leishmania strains, while meglumine antimoniate showed the lowest activity. Differences between amphotericin B vs other antileishmanial drug was statistically significant (P < 0·01).
Sensitivity of Leishmania EGFP-transfected strains to antileishmanial drugs
Data represent the median effective concentration (EC50) in μg mL−1 for each compound ± s.d.
a AMB: amphotericin B; MA: meglumine antimoniate; MIL: Miltefosine; PEN: pentamidine in Leishmania-EGFP strains.
*P < 0·01 AMB vs other drugs; **P < 0·01 L. panamensis vs other Leishmania-EGFP strains.
None of the EGFP-transfected Leishmania were responsive to meglumine antimonite, while the corresponding wild-type strain did respond. As seen by others sensitivity to meglumine antimoniate in wild-type strain varies among species being L. panamensis more sentitive to meglumine antomoniate while L. braziliensis showed the less sensitivity (Table 3).
Sensitivity of Leishmania wild-type strains to meglumine antimoniate
Data represent the median effective concentration (EC50) in μg mL−1 for meglumine antimoniate (MA) ± s.d. in Leishmania wild-type strains. *P < 0·01 L. panamensis vs other Leishmania strains.
DISCUSSION
In the present work, we compared some biological characteristics of L. braziliensis-EGFP, L. panamensis-EGFP, L. guyanensis-EGFP, L. mexicana-EGFP and L. amazonensis-EGFP that are species that cause cutaneous and mucosal leishmaniasis in the American region. Few studies have been conducted to understand the differences in growth kinetic, infective capability and drug sensitivity among Leishmania species and even fewer to determine if these biological criteria are affected by parasite transfection process. These studies are important to establish parameters to compare methodologies and variables included in biological assays used for in vitro studies of infectivity and antileishmanial activity.
This work was compared, simultaneously, with the behavior of five Leishmania-EGFP-transfected strains respect to the growth kinetic, infection capability and sensitivity to antileishmanial drugs. The in vitro growth of all the five Leishmania-EGFP strains was quite similar in terms of days to reach exponential or stationary growth. However, multiplication of L. braziliensis-EGFP and L. panamensis-EGFP was lower than that of L. mexicana-EGFP, L. amazonensis-EGFP and L. guyanensis-EGFP. Indeed, with comparable initial densities, there is a difference of about one log in growth between the time at which L. braziliensis-EGFP and L. panamensis-EGFP entered to stationary phase in comparison with L. mexicana-EGFP, L. amazonensis-EGFP and L. guyanensis-EGFP.
Interestingly, L. panamensis-EGFP behaves in the same way as does L. braziliensis-EGFP (both species belonging to Viannia subgenus) whereas L. guyanesis-EGFP (Viannia subgenus) display grown patterns similar to that L. amazonensis-EGFP and L. mexicana-EGFP, which belongs to Leishmania subgenus. Knowing the growth curve of parasites optimizes in vitro infection of macrophages. Here we found that the growth kinetics of the analysed species are similar with respect to time to late log phase occurs; moreover, the fifth day was detected as the ideal to do in vitro infection assays with any of these Leishmania strains. Recently, clinical isolates of L. braziliensis and L. amazonensis showed that metacyclogenesis occurs in 6 days old parasite cultures (Ovalle-Bracho et al. Reference Ovalle-Bracho, Franco-Muñoz, Londoño-Barbosa, Restrepo-Montoya and Clavijo-Ramírez2015).
Differences in infectivity between the EGFP strains were observed. One explanation could be that the number of infective parasites is different for each strain at the time of infection. Although early stationary phase parasites for all strains were used, the fraction of infective parasites may not be the same. Interestingly in the growth kinetics experiment L. braziliensis seem to reach stationary phase at a lower density and slightly earlier than the other strains and this strain has the lowest infective dose. This could easily be the result of a higher fraction of fully differentiated metacyclic parasites.
There are very few published work in which the infective capacity of Leishmania species using the in vitro infection of U937 cells was studied. However, in these papers a Leishmania:parasite cell ratio similar to that observed here was detected even that parasite used were from different strains. In those identified studies the infective parasite amount per U937 cell used was the following: 10:1 with L. amazonensis (LV78 strain) (Misra et al. Reference Misra, Tripathi and Chaudhuri2005; Da Silva et al. Reference Da Silva, Morato, Quixabeira, Pereira, Dorta, de Oliveira, Horta and Ribeiro-Dias2015), 15:1 (Ovalle-Bracho et al. Reference Ovalle-Bracho, Franco-Muñoz, Londoño-Barbosa, Restrepo-Montoya and Clavijo-Ramírez2015) or 10:1 (Da Silva et al. Reference Da Silva, Morato, Quixabeira, Pereira, Dorta, de Oliveira, Horta and Ribeiro-Dias2015) with L. braziliensis (M2903 strain), and 20:1 with L. panamensis (LS94 strain) (Puentes et al. Reference Puentes, Diaz, Hoya, Gutíerrez, Lozano, Patarroyo and Moreno2000), L. guyanensis (M4147 strain) (Puentes et al. Reference Puentes, Diaz, Hoya, Gutíerrez, Lozano, Patarroyo and Moreno2000) and L. mexicana (M379 strain) (Bertholet and Mauël, Reference Bertholet and Mauël2000).
Overall, the sensitivity of EGFP-transfected Leishmania strains to antileishmanial drugs did not show big variations among strains. All Leishmania-EGFP strains had high sensitivity to amphotericin B and pentamidine and moderate sensitivity to miltefosine. Only pentamidine and miltefosine did show a small difference in sensitivity between L. panamensis-EGFP in comparison with the other EGFP transfectant Leishmania strains. However, further studies are required to determine the basis of these differences.
Surprisingly, all EGFP-transfected Leishmania strains were not sensitive to meglumine antimoniate while the corresponding wild-type strains were sensitives. Because these experiments on sensitivity to meglumine antimoniate in EGFP transfectant and wild-type Leishmania strains were carried out using the same stock of meglumine antimoniate, this finding suggest that the insertion of the pIR3(-)-eGFP construct may affect the sensitivity of Leishmania to meglumine antimoniate.
Although the cellular mechanism of the antileishmanial activity of antimonials has not been fully understood, one posible explanation for the reduced sensitivity of GFP-expressing parasites to antimonials may be associated with the competition of the overexpressed eGFP with the SbIII, the active form of the drug, for the trypanothione reductase (TR), which play important roles in the intracellular redox system of the parasite. In this scenario, we propose that the overexpressed GFP could compete with SbIII for the main intracellular reduction system, the TR, rendering the TR inaccessible to SbIII and therefore reducing the toxic effects of this compound inside the cell.
It has been shown that SbIII inhibits the TR activity compromising the thiol-redox potential in promastigotes and amastigotes. On the other hand, it has been suggested that fluorescent proteins overexpression in bacteria and mammalian cells induce a myriad of physiological effects that impair the response to different antibiotics and compounds, respectively (Liu et al. Reference Liu, Jan, Chou, Chen and Ke1999; Allison and Sattenstall, Reference Allison and Sattenstall2007; Tao et al. Reference Tao, Evans, Yao, Cooper, Cornetta, Ballas, Hangoc and Broxmeyer2007). One of the proposed toxic mechanisms of GFP and other fluorescent proteins has been associated with the aggregation propensity of GFP inside the cell through intermolecular disulphide bond formation, where the protein aggregates may induce toxicity in the cell associated with oxidative stress. In order to preserve the auto-fluorescence and prevent cell toxicity the fluorescent protein should be reduced by non-described reduction systems inside the cell. As shown in our previous works and in the current report, GFP-transfected parasites are highly and homogeneously fluorescent implying an effective reduction process of the overexpressed protein inside the cell regardless the high transcription rate of the used promoter.
Concluding remarks
The in vitro growth kinetic, the number of promastigotes needed to infect the U937 cells and the level of sensitivity to a specific antileishmanial drug may varyin the Leishmania-EGFP strains. Moreover, the insertion of the pIR3(-)-eGFP may affect the sensitivity of the transfectant leishmanias to meglumine antimoniate and miltefosine but not to amphotericin B and pentamidine isethionate. In consequence, amphotericin B or pentamidine isethionate are the most adequated drugs to use as control use in vitro assays for antileishmanial activity, while neither meglumine antimoniate nor miltefosine should be used in in vitro assays as antileishmanial control drugs. Furthermore, during screening of drug candidates is necessary to test each compound against more than one Leishmania strain in order to verify that the antileihmanial activity of the tested compound is similar or not in those parasites. Interestingly the finding that GFP expressing parasites are refractory to antimonials activity may indicate a physiological alteration in the genetically modified parasites. It has been suggested that fluorescent proteins overexpression in bacteria and mammalian cells induce a myriad of physiological effects associated with the aggregation prone properties of fluorescent proteins through intermolecular disulfide bond formation. We hypothesize that TR may be involved in the reduction of the monomeric forms of eGFP in order to prevent its aggregation as can be inferred by the high and homogeneous fluorescence inside the parasites. However, simultaneous studies are needed to verify changes in the antileishmanial drug sensitivities of these transfected strains with respect to the correspondent wild-type strains.
ACKNOWLEDGEMENTS
To Dr. J. Clos, Bernard Notch Institute for Tropical Medicine, Hamburg-Germany who kindly donated the plasmid pIRmcs3(-) and J.F. Alzate, Universidad de Antioquia, Colombia, for his support during the development of the transgenic Leishmania strains.
FINANCIAL SUPPORT
The authors acknowledge support from Colciencias (CT-695-2014) and Universidad de Antioquia (sustainability program 2015).
CONFLICT OF INTEREST STATEMENT
The authors declare that there is no conflict of interests regarding the publication of this paper.
