Use of In Vivo and In Vitro Systems to SelectLeishmania amazonensisExpressing Green Fluorescent Protein

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-Leishmania 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, comparisons 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.

EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

“…Although there are some reporters about the toxic effect of EGFP expression in some cells (such as muscle (Wallace et al 2013), Ku80-deficient hamster cells (Koike et al 2013), retina (Rex et al 2004), mouse fibroblast, hamster kidney cell and Huh-7 cells from hepatoma cancer (Liu et al 1999), adult stem cells from rat hepatic (Taghizadeh and Sherley 2008), but recombinant parasites encoding reporters as EGFP have received great attention and are routinely used (Breton et al 2005;Costa Sdos et al 2011;Kamau et al 2001;Mehta et al 2008;Singh et al 2009;Varela M et al 2009) without adverse effects on parasite.…”

An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein

“…This could only be achieved with LaGFP after macrophage lysis as previously demonstrated using a spectrofluorimeter (Costa et al, 2011). One of the main advantages of using LaRFP is the elimination of the lysis step.…”

“…In Leishmania , GFP parasites have been developed for Leishmania major (Bolhassani et al, 2011; Kram et al, 2008; Mißlitz et al, 2000; Plock et al, 2001), Leishmania donovani , (Kaur et al, 2010; Singh and Dube, 2004; Singh et al, 2009) Leishmania infantum , (Bolhassani et al, 2011; Kamau et al, 2001) Leishmania mexicana (Mißlitz et al, 2000), and L. amazonensis (Boeck et al, 2006; Chan et al, 2003; Costa et al, 2011; Demicheli et al, 2004; Mehta et al, 2008; Mehta et al, 2010; Okuno et al, 2003). Although red fluorescent protein (RFP) parasites of L. major have been developed (Ng et al, 2008), no chemotherapeutic method is available using those transfectants.…”

Evaluation of the leishmanicidal and cytotoxic effects of inhibitors for microorganism metabolic pathway enzymes

“…Additionally, the relative intensity of fluorescence was analyzed in gfp-promastigotes incubated on black microplates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) as previously described [19]. The IC50 describes the drug concentrations that inhibit 50% of the promastigote proliferation and, was calculated using a curve fitting program (GraphPad Prism 6 software).…”

“…amazonensis (MHOM/BR/75/Josefa strain) isolated from a patient with cutaneous leishmaniosis in Brazil were transfected with green fluorescent protein (gfp) as previously described [19]. Promastigotes were cultured at 26 C in Earle 199 medium with 10% fetal calf serum [19].…”