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Infrared fluorescent imaging as a potent tool for in vitro, ex vivo and in vivo models of visceral leishmaniasis.

Calvo-Álvarez E, Stamatakis K, Punzón C, Álvarez-Velilla R, Tejería A, Escudero-Martínez JM, Pérez-Pertejo Y, Fresno M, Balaña-Fouce R, Reguera RM - PLoS Negl Trop Dis (2015)

Bottom Line: An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines.Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL.This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana, León, Spain.

ABSTRACT

Background: Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years.

Methodology/principal findings: We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks.

Conclusions/significance: The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

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Optimization of pLEXSY-iRFP vector by introducing downstream regions (DSR) of different genes.A) Schematic view of pLEXSY-hyg2 constructs containing infrared (iRFP) reporter gene and different DSR corresponding to A2, AMASTIN and HSP70-II genes. Constructs were linearized by SwaI digestion prior to electroporation. B) Correlation between fluorescence signal and the number of logarithmic promastigotes modified with iRFP-pLEXSY-HYG (□) or carrying the DSR of A2 (●), AMASTIN (○) or HSP70-II (Δ) genes. C) Flow-cytometry analysis of intracellular amastigotes isolated from THP-1 in vitro infections. D) Mean fluorescence intensity emitted by lesion-derived amastigotes obtained from WT and the engineered parasite strains carrying iRFP. Experiments were carried out with 107 parasites by triplicate and error bars represent standard deviations (SD). Significance level *** p< 0,001; ** p<0.01; from two tails of Student t-test.
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pntd.0003666.g002: Optimization of pLEXSY-iRFP vector by introducing downstream regions (DSR) of different genes.A) Schematic view of pLEXSY-hyg2 constructs containing infrared (iRFP) reporter gene and different DSR corresponding to A2, AMASTIN and HSP70-II genes. Constructs were linearized by SwaI digestion prior to electroporation. B) Correlation between fluorescence signal and the number of logarithmic promastigotes modified with iRFP-pLEXSY-HYG (□) or carrying the DSR of A2 (●), AMASTIN (○) or HSP70-II (Δ) genes. C) Flow-cytometry analysis of intracellular amastigotes isolated from THP-1 in vitro infections. D) Mean fluorescence intensity emitted by lesion-derived amastigotes obtained from WT and the engineered parasite strains carrying iRFP. Experiments were carried out with 107 parasites by triplicate and error bars represent standard deviations (SD). Significance level *** p< 0,001; ** p<0.01; from two tails of Student t-test.

Mentions: Once the iRFP reporter was chosen, we optimized the expression by replacing the original utr2 from pLEXSY-hyg2 vector with three sequences containing both 3’UTRs and IR (we will refer to as downstream regions, DSR). Two of these DSR belong to stage-regulated genes in the amastigote stage, the A2 and AMASTIN genes [23, 24]. The third DSR belongs to the HSP70 II gene of L. infantum, a sequence involved in mRNA stability at 37ºC [25]. Fig. 2A shows the schematic view of the different constructs containing the iRFP reporter gene.


Infrared fluorescent imaging as a potent tool for in vitro, ex vivo and in vivo models of visceral leishmaniasis.

Calvo-Álvarez E, Stamatakis K, Punzón C, Álvarez-Velilla R, Tejería A, Escudero-Martínez JM, Pérez-Pertejo Y, Fresno M, Balaña-Fouce R, Reguera RM - PLoS Negl Trop Dis (2015)

Optimization of pLEXSY-iRFP vector by introducing downstream regions (DSR) of different genes.A) Schematic view of pLEXSY-hyg2 constructs containing infrared (iRFP) reporter gene and different DSR corresponding to A2, AMASTIN and HSP70-II genes. Constructs were linearized by SwaI digestion prior to electroporation. B) Correlation between fluorescence signal and the number of logarithmic promastigotes modified with iRFP-pLEXSY-HYG (□) or carrying the DSR of A2 (●), AMASTIN (○) or HSP70-II (Δ) genes. C) Flow-cytometry analysis of intracellular amastigotes isolated from THP-1 in vitro infections. D) Mean fluorescence intensity emitted by lesion-derived amastigotes obtained from WT and the engineered parasite strains carrying iRFP. Experiments were carried out with 107 parasites by triplicate and error bars represent standard deviations (SD). Significance level *** p< 0,001; ** p<0.01; from two tails of Student t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380447&req=5

pntd.0003666.g002: Optimization of pLEXSY-iRFP vector by introducing downstream regions (DSR) of different genes.A) Schematic view of pLEXSY-hyg2 constructs containing infrared (iRFP) reporter gene and different DSR corresponding to A2, AMASTIN and HSP70-II genes. Constructs were linearized by SwaI digestion prior to electroporation. B) Correlation between fluorescence signal and the number of logarithmic promastigotes modified with iRFP-pLEXSY-HYG (□) or carrying the DSR of A2 (●), AMASTIN (○) or HSP70-II (Δ) genes. C) Flow-cytometry analysis of intracellular amastigotes isolated from THP-1 in vitro infections. D) Mean fluorescence intensity emitted by lesion-derived amastigotes obtained from WT and the engineered parasite strains carrying iRFP. Experiments were carried out with 107 parasites by triplicate and error bars represent standard deviations (SD). Significance level *** p< 0,001; ** p<0.01; from two tails of Student t-test.
Mentions: Once the iRFP reporter was chosen, we optimized the expression by replacing the original utr2 from pLEXSY-hyg2 vector with three sequences containing both 3’UTRs and IR (we will refer to as downstream regions, DSR). Two of these DSR belong to stage-regulated genes in the amastigote stage, the A2 and AMASTIN genes [23, 24]. The third DSR belongs to the HSP70 II gene of L. infantum, a sequence involved in mRNA stability at 37ºC [25]. Fig. 2A shows the schematic view of the different constructs containing the iRFP reporter gene.

Bottom Line: An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines.Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL.This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana, León, Spain.

ABSTRACT

Background: Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years.

Methodology/principal findings: We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks.

Conclusions/significance: The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

Show MeSH
Related in: MedlinePlus