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A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit.

De Muylder G, Ang KK, Chen S, Arkin MR, Engel JC, McKerrow JH - PLoS Negl Trop Dis (2011)

Bottom Line: In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage.Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite.This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sandler Center for Drug Discovery, University of California San Francisco, San Francisco, California, United States of America. Geraldine.DeMuylder@ucsf.edu

ABSTRACT
The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

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Infection of THP-1 with L. donovani: detection, segmentation and growth of host cell and parasite.A–D. Detection and segmentation of THP-1 host cell and L. donovani intracellular amastigotes. Images obtained with the INCell Analyzer 1000 (20X) of THP-1 cells infected with L. donovani and treated with 1% DMSO (A, B) or 2 µM amphotericin B (C, D). Insert shows the relative fluorescence of DAPI-stained parasite kinetoplast (k) and nucleic DNA (n) and host cell nucleus (N). Segmentation of host cell nuclei and parasite kinetoplast using INCell developer toolbox software (B, D). Red outline: parasite kinetoplast, blue outlines: host cell nucleus and border representing the boundary of the host cell. E. Evolution of the number of parasites and THP-1 host cells in a 72 h time course. THP-1 and L. donovani were counted at several time points after infection using the INCell 1000. White squares: average number of host nuclei per well (n = 8); black circles: average number of parasites counted per well divided by the total number of host nuclei per well (n = 8).
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pntd-0001253-g001: Infection of THP-1 with L. donovani: detection, segmentation and growth of host cell and parasite.A–D. Detection and segmentation of THP-1 host cell and L. donovani intracellular amastigotes. Images obtained with the INCell Analyzer 1000 (20X) of THP-1 cells infected with L. donovani and treated with 1% DMSO (A, B) or 2 µM amphotericin B (C, D). Insert shows the relative fluorescence of DAPI-stained parasite kinetoplast (k) and nucleic DNA (n) and host cell nucleus (N). Segmentation of host cell nuclei and parasite kinetoplast using INCell developer toolbox software (B, D). Red outline: parasite kinetoplast, blue outlines: host cell nucleus and border representing the boundary of the host cell. E. Evolution of the number of parasites and THP-1 host cells in a 72 h time course. THP-1 and L. donovani were counted at several time points after infection using the INCell 1000. White squares: average number of host nuclei per well (n = 8); black circles: average number of parasites counted per well divided by the total number of host nuclei per well (n = 8).

Mentions: The human leukemia monocyte cell line THP-1 has been commonly used as a model for Leishmania infection and has been described as a suitable model for drug screening [24], [25]. In vitro infection of macrophages by Leishmania and analysis of intracellular parasite growth requires a method allowing for robust detection, discrimination and counting of parasites and host cells. In our setting, THP-1 cells infected with L. donovani were stained with the DNA marker DAPI (4′,6′-diamidino-2-phenylindole) allowing the visualization of host cell nuclei and parasite kinetoplasts. Images collected with an INCell Analyzer 1000 fluorescent microscope showed a significant size difference between host cell nuclei and parasite kinetoplasts. This feature was exploited for image segmentation and determination of the number of host cells and parasites (Figure 1A–D). The ratio between total number of parasites and total number of host cells was calculated for each well. In addition, counts of host cell nuclei were used as a quantitative measure of cell toxicity induced by the compounds. Incubation of L. donovani with THP-1 for 4 hours at a ratio of 15 parasites per host cell led to an average infection of 4.1 +/− 0.32 parasites per host cell after 72 h incubation, with an average of 30 +/− 9 percent of the cells infected and no change in the number of host cells (Figure 1E). Growth of parasite and host cells was not affected by 1% DMSO (Figure 2A–B). Amphotericin B, the first line drug used against leishmaniasis, was used as a positive control. At 2 µM amphotericin B did not affect THP-1 host macrophages (Figure 2A) but significantly inhibited growth of intracellular L. donovani (Figure 2B) with an estimated GI50 of 0.08 µM (Figure 2C). This is comparable to GI50 values from previous reports [11], [20].


A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit.

De Muylder G, Ang KK, Chen S, Arkin MR, Engel JC, McKerrow JH - PLoS Negl Trop Dis (2011)

Infection of THP-1 with L. donovani: detection, segmentation and growth of host cell and parasite.A–D. Detection and segmentation of THP-1 host cell and L. donovani intracellular amastigotes. Images obtained with the INCell Analyzer 1000 (20X) of THP-1 cells infected with L. donovani and treated with 1% DMSO (A, B) or 2 µM amphotericin B (C, D). Insert shows the relative fluorescence of DAPI-stained parasite kinetoplast (k) and nucleic DNA (n) and host cell nucleus (N). Segmentation of host cell nuclei and parasite kinetoplast using INCell developer toolbox software (B, D). Red outline: parasite kinetoplast, blue outlines: host cell nucleus and border representing the boundary of the host cell. E. Evolution of the number of parasites and THP-1 host cells in a 72 h time course. THP-1 and L. donovani were counted at several time points after infection using the INCell 1000. White squares: average number of host nuclei per well (n = 8); black circles: average number of parasites counted per well divided by the total number of host nuclei per well (n = 8).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139667&req=5

pntd-0001253-g001: Infection of THP-1 with L. donovani: detection, segmentation and growth of host cell and parasite.A–D. Detection and segmentation of THP-1 host cell and L. donovani intracellular amastigotes. Images obtained with the INCell Analyzer 1000 (20X) of THP-1 cells infected with L. donovani and treated with 1% DMSO (A, B) or 2 µM amphotericin B (C, D). Insert shows the relative fluorescence of DAPI-stained parasite kinetoplast (k) and nucleic DNA (n) and host cell nucleus (N). Segmentation of host cell nuclei and parasite kinetoplast using INCell developer toolbox software (B, D). Red outline: parasite kinetoplast, blue outlines: host cell nucleus and border representing the boundary of the host cell. E. Evolution of the number of parasites and THP-1 host cells in a 72 h time course. THP-1 and L. donovani were counted at several time points after infection using the INCell 1000. White squares: average number of host nuclei per well (n = 8); black circles: average number of parasites counted per well divided by the total number of host nuclei per well (n = 8).
Mentions: The human leukemia monocyte cell line THP-1 has been commonly used as a model for Leishmania infection and has been described as a suitable model for drug screening [24], [25]. In vitro infection of macrophages by Leishmania and analysis of intracellular parasite growth requires a method allowing for robust detection, discrimination and counting of parasites and host cells. In our setting, THP-1 cells infected with L. donovani were stained with the DNA marker DAPI (4′,6′-diamidino-2-phenylindole) allowing the visualization of host cell nuclei and parasite kinetoplasts. Images collected with an INCell Analyzer 1000 fluorescent microscope showed a significant size difference between host cell nuclei and parasite kinetoplasts. This feature was exploited for image segmentation and determination of the number of host cells and parasites (Figure 1A–D). The ratio between total number of parasites and total number of host cells was calculated for each well. In addition, counts of host cell nuclei were used as a quantitative measure of cell toxicity induced by the compounds. Incubation of L. donovani with THP-1 for 4 hours at a ratio of 15 parasites per host cell led to an average infection of 4.1 +/− 0.32 parasites per host cell after 72 h incubation, with an average of 30 +/− 9 percent of the cells infected and no change in the number of host cells (Figure 1E). Growth of parasite and host cells was not affected by 1% DMSO (Figure 2A–B). Amphotericin B, the first line drug used against leishmaniasis, was used as a positive control. At 2 µM amphotericin B did not affect THP-1 host macrophages (Figure 2A) but significantly inhibited growth of intracellular L. donovani (Figure 2B) with an estimated GI50 of 0.08 µM (Figure 2C). This is comparable to GI50 values from previous reports [11], [20].

Bottom Line: In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage.Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite.This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sandler Center for Drug Discovery, University of California San Francisco, San Francisco, California, United States of America. Geraldine.DeMuylder@ucsf.edu

ABSTRACT
The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

Show MeSH
Related in: MedlinePlus