357 © 2011, Korean Society for Parasitology This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. INTRODUCTION Advances in genetic manipulation with transfection tech- niques permit the preparation of Leishmania expressing report- er genes, such as green fluorescent protein (GFP) from jellyfish Aequorea victoria [1-9]. In general, GFP-based methods are sim- pler and easier to perform in studies of cellular dynamics than non-reporter and other reporter-gene-based methods and can be performed in living samples [10,11]. Various Leishmania spe- cies have been transfected with GFP using episomal vectors and then maintained under selective pressure in vitro using antibi- otics, including aminoglycoside geneticin sulphate (G418) [1,4]. Most reports of GFP-Leishmania have used the flagellated extra- cellular promastigote, the stage of parasite detected in the mid- gut of the sandfly vector and which is absent in the mammali- an host [1,4-6,8,9,12], however, few studies have been per- formed with amastigotes, the stage of parasite exclusively de- tected in mammals [2,3]. The importance lies in the fact that amastigotes reside in macrophages and are the target for che- motherapy and drug screening tests [9,13]. In this study, comparisons were made regarding the efficien- cy for in vitro G418 selection of GFP-L. amazonensis promasti- gotes and amastigotes, a species that causes cutaneous and cu- taneous metastatic lesions in South American countries [14,15]. For the first time, the use of the in vivo G418 selection for GFP- Leishmania amastigotes is also described. MATERIALS AND METHODS Parasites Leishmania amazonensis (MHOM/BR/75/Josefa strain) were rendered fluorescent as previously described [1,16]. Briefly, promastigotes were transfected by electroporation with the gene fragment coding for the C-terminal extension of cysteine proteinase fused to the reporter GFP in the pXG-́ GFP+ vector, a derivative of pX63NEO, developed by Ha and coworkers [1]. Promastigotes were cultured at 26 C̊ in Earle 199 medium with 10% fetal calf serum (FCS) (Cultilab, Campinas São Paulo, Bra- ISSN (Print) 0023-4001 ISSN (Online) 1738-0006 Korean J Parasitol Vol. 49, No. 4: 357-364, December 2011 http://dx.doi.org/10.3347/kjp.2011.49.4.357 Use of In Vivo and In Vitro Systems to Select Leishmania amazonensis Expressing Green Fluorescent Protein Solange dos Santos Costa1, Marjorie de Assis Golim2, Bartira Rossi-Bergmann3, Fabio Trindade Maranhão Costa4 and Selma Giorgio1,* 1Department of Animal Biology, Biology Institute, Universidade Estadual de Campinas Caixa Postal 6109, Cep 13.083-970, Campinas, São Paulo, Brazil; 2Department of Genetics, Evolution and Bioagents, Biology Institute, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil; 3Blood Center, Faculty of Medicine, School of Medicine, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil; 4Carlos Chagas Filho Institute, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Abstract: Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishma- nia have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, compari- sons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions. Key words: Leishmania amazonensis, green fluorescent protein, macrophage, geneticin •Received 4 July 2011, revised 9 August 2011, accepted 24 August 2011. *Corresponding author (sgiorgio@unicamp.br) 358 Korean J Parasitol Vol. 49, No. 4: 357-364, December 2011 zil), and amastigotes were isolated from footpad lesions of in- fected BALB/c mice, as previously described [17]. The experi- mental protocols were approved by the Animal Ethics Com- mittee of the Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. Selection of GFP-L. amazonensis G418 was purchased from Sigma (Sigma, St. Louis, Missouri, USA) and diluted in water at 50 mg/ml. For in vitro selection, GFP-promastigotes and GFP-amastigotes were cultured in 25 cm2 culture flasks and exposed to different concentrations of G418. Untreated parasites were maintained for each assay. For in vivo selection, BALB/c mice were inoculated with 2×107 pro- mastigotes (70-80% GFP-promastigotes positives) subcutane- ously in the hind footpad, and at different times postinfection, mice received daily intralesional dose of G418 (40 mg/kg of body weight/day). For each group, mice were administered 3, 5, 6, or 7 doses of G418 or saline only. Control mice received intralesional injections with saline solution. Cultured promas- tigotes and amastigotes, and amastigotes isolated from lesions, were analyzed by flow cytometry [17]. The parasites were visu- alized under a Nikon Eclipse 50i fluorescence microscope (Ni- kon, Tokyo, Japan). All images were captured and analyzed with a digital camera (Nikon DXM1200-F) and imaging soft- ware (ACT-1, Nikon). The parasite count was determined by optical microscopy. Flow cytometry The GFP-promastigotes and GFP-amastigotes (2×106) were fixed in 1% formaldehyde and washed in PBS. GFP expression was analyzed by flow cytometer FACSCalibur (Becton Dickin- son Biosciences, Franklin, New Jersey, USA), and the parasite population was selected using the parameters of size (Forward Scatter-FSC) versus internal complexity (Side Scatter-SSC), us- ing a logarithmic scale. Spectrofluorometric analyses The GFP-promastigotes and GFP-amastigotes were serially diluted and incubated on black microplates (Nunc, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The corre- spondence between the relative intensity of fluorescence to the cell count was analyzed using a spectrofluorimeter (Multi-De- tection Microplate Reader- Synergy HT, Bio-Tek, Winooski, Vermont, USA) at 485 nm excitation and 528 nm emission [1,18]. Fluorescence is reported in arbitrary fluorescence units and the data represent GFP fluorescence of parasites subtracted out control wells (saline). The wild type L. amazonensis emit- ted undetectable levels of GFP fluorescence. Macrophage infection with GFP-L. amazonensis J774 macrophages or peritoneal macrophages from normal BALB/c mice, obtained as previously described [19], were cul- tured in Earle 199 medium with 10% FCS. Cells were infected with GFP-promastigotes at a parasite cell ratio of 10:1 for 24 hr at 34˚C. After removal of extracellular parasites, the infected cell cultures were incubated at 37˚C for 24 hr and the intracel- lular forms observed microscopically, as previously described [19]. Cell cultures were also fixed in 1% formaldehyde diluted in 300 μl PBS at a density of 2×106 and analyzed by flow cy- tometry. For spectrofluorometric analyses, cell cultures were lysed with distilled water and transferred to black microplates to determine fluorescence. Data analyses Experiments were repeated at least 3 times and the results are expressed as the mean±SD. The results presented were ver- ified in 3 mice per treatment. Control mice were treated with saline. Data were analyzed by 1-way ANOVA and the Student’s t-test (P<0.01). RESULTS In vitro G418 selection of GFP-L. amazonensis promastigotes As mentioned previously, promastigotes are currently used for GFP transfection experiments [1-8]. The period of episom- al plasmid retention and efficiency of G418 selection were an- alyzed in L. amazonensis GFP-promastigotes. As seen in Fig. 1A, the fluorescence intensity was proportional to the number of parasites when serial dilutions of promastigotes cultured for 7, 14, 32, and 41 days without G418 were conducted. When GFP- promastigotes were cultured for 56 days without G418, the fluorescence intensity was not proportional to the number of promastigotes (Fig. 1A), indicating a low fluorescence signal from parasites cultured for this period. Flow cytometry analy- sis (Fig. 1C) verified that the percentage of GFP-promastigotes decreased with time, and 80.7%, 42.8%, 27.4%, 17.9% para- sites were GFP-positive after 60, 90, 120, and 210 days without G418 treatment, respectively. In fact, when promastigotes were treated with 1 mg/ml G418, 94.2% were GFP-positive after 5 Costa et al.: In vitro and in vivo selection of GFP from Leishmania amazonensis 359 days (Fig. 1C), the fluorescence intensity was high and propor- tional to the number of parasites (Fig. 1B) and death of G418 non-resistant promastigotes occurred (data not shown). Bright- ly fluorescence GFP-promastigotes presenting normal morphol- ogy were observed by microscopy (Fig. 1D). Taken together, these results indicate that the fluorescence intensity and the percentage of GFP-positive promastigotes decreased after 56- 60 days without G418 selection. In addition, G418 treatment was necessary and efficient for GFP-promastigote selection. In vitro G418 selection of L. amazonensis GFP-amastigotes The results of GFP-amastigotes in vitro selection is shown in Fig. 2. The amastigotes were taken from mouse lesions and cultured with G418. The number of parasites decreased with time of G418 treatment; after 19 hr, death was induced in 34%, 42%, and 62% of amastigotes treated with 200 µg/ml, 500 µg/ ml, and 1 mg/ml, respectively (Fig. 2A). The fluorescence in- tensity was proportional to the number of parasites when seri- al dilutions were conducted on GFP-amastigotes treated with G418, 500 µg/ml and 1 mg/ml (Fig. 2B). Although a small population of GFP-positive amastigotes (20%) was detected by flow cytometry in parasites treated with G418, 1 mg/ml (Fig. 2C), very bright fluorescence was observed in these amasti- gotes (Fig. 2D). A comparison of results (Fig. 2) with GFP-pro- mastigotes selection (Fig. 1) indicated that in vitro G418 selec- tion was less effective with amastigotes. 50 45 40 35 30 25 20 15 10 5 0 Fl uo re sc en ce 1,00E+05 1,00E+06 Parasite number 1,00E+07 Parasite number 300 250 200 150 100 50 0 Fl uo re sc en ce 1,00E+05 1,00E+06 1,00E+07 A B C D 200 160 120 80 40 0 200 160 120 80 40 0 100 101 102 103 104 FL1-Height 100 101 102 103 104 FL1-Height 80.7% 42.8% M1 M1 200 160 120 80 40 0 100 101 102 103 104 FL1-Height M1 94.2% C1 200 160 120 80 40 0 100 101 102 103 104 FL1-Height M1 27.4% C4 200 160 120 80 40 0 100 101 102 103 104 FL1-Height M1 17.9% C5 100 80 60 40 20 0 100 101 102 103 104 FL1-Height M1 1.6% C6 C2 C3 D1 D2 D4 D3 10 µm 10 µm 10 µm 10 µm Fig. 1. GFP-L. amazonensis promastigote fluorescence analysis. (A) The fluorescence signal plotted against promastigote counts. Data at 7 days (♦); 14 days (■); 32 days (▲); 41 days (X) and 56 days of parasite culture (●). (B) The fluorescence signal plotted against the number of promastigotes treated with 100 µg/ml (♦), 150 µg/ml (■) and 1 mg/ml (▲) of G418 or untreated (X). Fluorescence is re- ported in arbitrary fluorescence units. (C) Flow cytometry analyses of GFP-promastigotes 5 days (C1), 60 days (C2), 90 days (C3), 120 days (C4), 210 days (C5) after G418 selection and wild L. amazonensis promastigotes (C6). (D) Microscopic images of GFP-pro- mastigotes. Phase contrast images of G418 selected GFP-promastigotes (D1) and non-selected GFP-promastigotes (D3). Fluores- cence images of G418 selected GFP-promastigotes (D2) and non-selected GFP-promastigotes (D4). Magnification 400× . 360 Korean J Parasitol Vol. 49, No. 4: 357-364, December 2011 In vivo G418 selection of L. amazonensis GFP-amastigotes Since amastigotes are the intracellular stage in mammalian hosts and show an exuberant growth in the lesions induced by L. amazonensis in susceptible mice [17], in vivo selection of GFP-amastigotes was evaluated. Mice were infected with GFP- promastigotes and intralesional injections were administered at different postinfection times (Fig. 3). It should be noted that the stability of GFP expression in amastigotes was low, since only 24.0%, 20.4-23.3%, and 11.9-14.0% of the lesion deriv- ed-amastigotes from untreated G418 mice were GFP-positive after 15, 30, and 90 days of infection, respectively (Fig. 3). Most of the treatment courses resulted in a modest increase in GFP- positive amastigotes; for example, administration of 5 doses of G418 after 30 days postinfection compared with saline treat- ment, resulted in 41.4% positive GFP-positive vs 23.3% GFP- positive amastigotes, respectively (Fig. 3C, D). Another treat- ment was administered, increasing the number of G418 doses to 6, and resulted in more efficient selection of GFP-amasti- gotes (30.2% GFP-positive amastigotes isolated from lesions G418-treated vs 11% GFP-positive amastigotes from lesions treated with saline); a 3-fold increase in the number of GFP- positive amastigotes (Fig. 3I, J). In vitro infection with GFP-L. amazonensis To examine the ability of G418 selected parasites to infect cells, the infection level and fluorescence signal of macrophage cultures were evaluated. As shown in Fig. 4A, the infection lev- el in murine peritoneal macrophages incubated with GFP-pro- Fig. 2. GFP-L. amazonensis amastigote fluorescence analysis. (A) The number of GFP-amastigotes in vitro treated with 200 µg/ml (♦), 500 µg/ml (▲) and 1 mg/ml (■) of G418. (B). The fluorescence signal plotted against the number of amastigotes cultured in vitro treat- ed with 200 µg/ml (♦), 500 µg/ml (■) and 1 mg/ml (▲) of G418. Fluorescence is reported in arbitrary fluorescence units. (C) Flow cy- tometry analyses of wild L. amazonensis amastigotes (C1), non-selected GFP-amastigotes (C2) and G418 selected GFP-amastigotes (C3). (D) Microscopic images of GFP-amastigotes. Phase contrast images of non-selected GFP-amastigotes (D2); 200 µg/ml G418 selected GFP-amastigotes (D4); 500 µg/ml G418 selected GFP-amastigotes (D6); 1 mg/ml G418 selected GFP-amastigotes (D8); Fluorescence images of non-selected GFP-amastigotes (D1); 200 µg/ml G418 selected GFP-amastigotes (D3); 500 µg/ml G418 se- lected GFP-amastigotes (D5); 1 mg/ml G418 selected GFP-amastigotes (D7). Magnification 400× . 4,00E+07 3,50E+07 3,00E+07 2,50E+07 2,00E+07 1,50E+07 1,00E+07 5,00E+06 0,00E+00 Pa ra si te n um be r/m l 0 2 4 19 Hours A 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Fl uo re sc en ce 1,00E+06 1,00E+07 1,00E+08 Parasite number B C D 150 120 90 50 30 0 100 101 102 103 104 FL1-Height M1 1.3% C1 C ou nt s 150 120 90 50 30 0 100 101 102 103 104 FL1-Height M1 7.4% C2 C ou nt s 150 120 90 50 30 0 100 101 102 103 104 FL1-Height M1 20% C3 C ou nt s D1 D2 D5 D6 D3 D4 D7 D8 6 µm 6 µm 6 µm 6 µm 6 µm 6 µm 6 µm 6 µm Costa et al.: In vitro and in vivo selection of GFP from Leishmania amazonensis 361 mastigotes was high, around 70% of infected cells and 3 amas- tigotes per cell. The GFP-parasite infected macrophages also revealed a strong fluorescence signal (Fig. 4B) and using flow cytometry, a high percentage of GFP-positive macrophages were detected after incubation with GFP-promastigotes (95.1%) (Fig. 4D). Light and fluorescence microscopy demonstrated the ability of GFP-L. amazonensis to infect macrophages (Fig. 4C). Similar results were obtained when macrophages were infected with GFP-amastigotes and the J774 cell line was used instead of peritoneal macrophages (data not shown). The in vitro and in vivo infectivities of GFP-L. amazonensis were simi- lar to those of wild L. amazonensis for both promastigotes or amastigotes (data not shown). DISCUSSION Although many studies have used GFP-Leishmania to screen antileishmanial compounds and analyze parasite-host interac- Fig. 3. Flow cytometry analyses of GFP-amastigotes after in vivo G418 selection. Flow cytometry of murine lesion derived GFP-amastigotes after: 15 days infection and 7 doses of saline treatment (A); 15 days infection and 7 doses of G418 treat- ment (B); 30 days infection and 5 doses of saline treatment (C); 30 days infection and 5 doses of G418 treatment (D); 30 days infection and 9 doses of saline treat- ment (E); 30 days infection and 9 doses of G418 treatment (F); 90 days infection and 3 doses of saline treatment (G); 90 days infection and 3 doses of G418 treatment (H); 90 days infection and 6 doses of saline treatment (I); 90 days infection and 6 doses of G418 treatment (J). G418 and saline were administered as described in Materials and Methods. 150 120 90 60 30 0 100 101 102 103 104 FL1-Height M1 24% C ou nt s 200 160 120 80 40 0 100 101 102 103 104 FL1-Height M1 41.4% C ou nt s 100 80 60 40 20 0 100 101 102 103 104 FL1-Height M1 14% C ou nt s 100 80 60 40 20 0 100 101 102 103 104 FL1-Height M1 30.2% C ou nt s 100 80 60 40 20 0 100 101 102 103 104 FL1-Height M1 21% C ou nt s 100 80 60 40 20 0 100 101 102 103 104 FL1-Height M1 11.9 C ou nt s 200 160 120 80 40 0 100 101 102 103 104 FL1-Height M1 20.4% C ou nt s 200 160 120 80 40 0 100 101 102 103 104 FL1-Height M1 39.7% C ou nt s 150 120 90 60 30 0 100 101 102 103 104 FL1-Height M1 34.5% C ou nt s 200 160 120 80 40 0 100 101 102 103 104 FL1-Height M1 23.3% C ou nt s A B C D E F G J H I 362 Korean J Parasitol Vol. 49, No. 4: 357-364, December 2011 tion mechanisms, current understanding of G418 selective pres- sure on promastigotes and amastigotes is poor [1-9]. The ten- dency to silence transgene expression over time is a character- istic of transgenic cells and consequently, antibiotic selection results in a proliferative advantage for the subset of cells ex- pressing antibiotic resistance gene [10]. In this study, monitor- ing the stability of GFP expression over a period of 210 days indicated fluorescence decreased in promastigotes cultured 60 days without G418, i.e., GFP-promastigotes retained episomal plasmid for a prolonged period. Reports have referred to the β-galactosidase activity of recombinant L. amazonensis promas- tigotes for 30 days without selective pressure [6] and to the episomal expression of GFP in hamster ovary cells transfected with GFP and cultured 24 days without G418 [20]. The stabili- ty of GFP expression in amastigotes is low, since only 24% of amastigotes derived from murine lesions were GFP-positive af- ter 15 days of infection. The reasons contributing to low reten- tion of episomal plasmid in amastigotes remain unknown; epi- genetic effects, such as DNA methylation, have been implicat- ed in this phenomenon in some mammalian cell lines [21] and need to be evaluated in Leishmania. Next, G418 selection for GFP-L. amazonensis was evaluated. Analysis of the results indicated that amastigote ability to be selected for GFP expression in vitro was poor compared with promastigotes. The best results were obtained with 1 mg/ml G418 (20% of positive GFP-amastigotes vs 94% of positive GFP-promastigotes). Following these experiments, our group evaluated whether it was possible to select GFP-amastigotes in vivo using mice lesions. Such a selection system would be use- ful in circumstances in which it is not possible to use an in vi- tro selection system. Again, analysis of the results indicated that amastigote ability to be selected in vivo was poor (the per- centage of positive GFP-amastigotes reached a maximum of 41.4%), indicating that G418 selection is not effective with this parasite form. No report of the use of in vivo systems to test G418 selection in Leishmania is available for comparison with 2 1 0 20 40 60 80 100 2 4 6 8 Amastigote/macrophage % Infected macrophages 80,000 70,000 60,000 50,000 40,000 30,000 20,000 10,000 0 Fl uo re sc en ce Control Infected macrophages A B C1 D1 D2 D3 C2 C3 200 160 120 80 40 0 100 101 102 103 104 FL1-Height M1 1.2% C ou nt s D1 200 160 120 80 40 0 100 101 102 103 104 FL1-Height M1 95.1% C ou nt s D2 C D Fig. 4. Infection of murine macrophages with GFP-L. amazonensis. (A) Number of amastigotes per macrophage and % of infected macrophages after infection with GFP-promastigotes. (B) The fluorescence signal of noninfected macrophages (control) and infected macrophages. Fluorescence is reported in arbitrary fluorescence units. (C) Microscopic images of infected macrophages. Fluores- cence image (C1); Phase contrast image (C2) and Giemsa stained image (C3). Magnification 400× . (D) Flow cytometry of noninfect- ed macrophages (D1) and infected macrophages (D2). Costa et al.: In vitro and in vivo selection of GFP from Leishmania amazonensis 363 our results; however, Murphy and coworkers [22] selected high numbers of transfected Trypanosoma brucei inoculating G418 in infected mice and concluded that the extracellular trypanosome blood form was highly sensitive to G418. The reasons for the high level of sensitivity of T. brucei trypanosome compared to L. amazonensis amastigote are unclear, but may be due to the prob- lem of intracellular antibiotic delivery to the parasite and/or the acidic pH of the parasitophorous vacuoles housing amasti- gotes, since G418 selection is pH-dependent [23]. In conclusion, analysis of the results of the present study showed that L. amazonensis GFP-promastigotes retained epi- somal plasmid for a prolonged period (i.e., 60 days) and G418 treatment is necessary and efficient for GFP-promastigote se- lection. In addition, the analyses of amastigotes showed that the intracellular parasite presented low retention of episomal plasmid in the absence of G418 selection and low levels of sensitivity to G418. The development of high level regulatable expression systems to boost transgenic protein levels in Leish- mania amastigotes will be essential to advance the studies of infection dynamics in vitro and in vivo. ACKNOWLEDGMENTS This work was supported by the Fundação de Amparo à Pes- quisa do Estado de São Paulo, Coordenação de Aperfeiçoamen- to de Pessoal de Nível Superior and the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil. REFERENCES 1. Ha DS, Schwarz JK, Turco SJ, Beverley SM. 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