Skip to main content
main-content

01.12.2012 | Research | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

ATP and luciferase assays to determine the rate of drug action in in vitro cultures of Plasmodium falciparum

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Tasmiyah Khan, Anna C van Brummelen, Christopher J Parkinson, Heinrich C Hoppe
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-369) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

HCH and CJP conceived and designed the study, TK and ACvB performed the experimental procedures and data analysis, HCH and CJP were principally responsible for interpretation of the data. HCH and TK drafted the manuscript, which was read, amended and approved by all authors. All authors read and approved the final manuscript.

Abstract

Background

Knowledge of the rate of action of compounds against cultured malaria parasites is required to determine the optimal time-points for drug mode of action studies, as well as to predict likely in vivo parasite clearance rates in order to select optimal hit compounds for further development. In this study, changes in parasite ATP levels and transgenic luciferase reporter activity were explored as means to detect drug-induced stress in cultured parasites.

Methods

In vitro cultures of Plasmodium falciparum 3D7 wild-type or firefly luciferase-expressing parasites were incubated with a panel of six anti-malarial compounds for 10 hours and parasite ATP levels or luciferase activity determined at two-hour intervals using luminescence-based reagents. For comparative purposes, parasite morphology changes were evaluated by light microscopy, as well as the extent to which parasites recover after 48 hours from a six-hour drug treatment using a parasite lactate dehydrogenase assay.

Results

Changes in parasite ATP levels displayed three phenotypes: mild or no change (chloroquine, DFMO); 2–4 fold increase (mefloquine, artemisinin); severe depletion (ritonavir, gramicidin). The respective phenotypes and the rate at which they manifested correlated closely with the extent to which parasites recovered from a six-hour drug treatment (with the exception of chloroquine) and the appearance and severity of morphological changes observed by light microscopy. Luciferase activity decreased profoundly in parasites treated with mefloquine, artemisinin and ritonavir (34-67% decrease in 2 hours), while chloroquine and DFMO produced only mild changes over 10 hours. Gramicidin yielded intermediate decreases in luciferase activity.

Conclusions

ATP levels and luciferase activity respond rapidly to incubation with anti-malarial drugs and provide quantitative read-outs to detect the appearance and magnitude of drug-induced stress in cultured parasites. The correlation between the observed changes and irreversible parasite toxicity is not yet sufficiently clear to predict clinical clearance rates, but may be useful for ranking compounds against each other and standard drugs vis-à-vis rate of action and for determining early time-points for drug mode of action studies.
Zusatzmaterial
Additional file 1: Figure A1. Correlation of ATP luminescence signals with parasite numbers and stages. A. Aliquots were removed from a trophozoite-stage culture and the parasites isolated. Serial dilutions of the parasites were performed in PBS and ATP levels measured. Parasite numbers were calculated from the percentage parasitaemia and red blood cell concentration (determined with a haemocytometer) of the original culture. Three samples were processed in parallel. Note that the lowest parasite number (1.23x105) corresponds to approximately 4μl of a 2% parasitaemia, 5% haematocrit culture (or 0.2μl packed red blood cells) and produced an average luminescence reading of 7030. B. Ring-stage parasites were isolated from three 0.5ml aliquots of a sorbitol synchronized 5% haematocrit 10% parasitaemia culture and snap-frozen in liquid nitrogen. After 24h, additional 0.5ml aliquots were removed from the same culture and used to prepare frozen trophozoite-stage parasites. After thawing, ATP luminescence signals in the samples were determined and compared to background signals obtained with aliquots of uninfected red blood cell cultures processed in parallel. The trophozoite samples produced a mean luminescence reading of 354054, compared 13121 for rings and 993 for uninfected red blood cells (note the log scale of the Y-axis). (PDF 176 KB)
12936_2012_2509_MOESM1_ESM.pdf
Additional file 2: Table 1. IC50 values of compounds used in this study. IC50 values were obtained by incubating P. falciparum 3D7 cultures with serial dilutions of the test compounds for 48 hours and assessing parasite viability using the parasite lactate dehydrogenase (pLDH) assay. Values are shown as averages ± standard deviation for three independent determinations. IC50 values for lactacystin and MG-132 were determined on a single occasion against luciferase-expressing parasites. (PDF 48 KB)
12936_2012_2509_MOESM2_ESM.pdf
Additional file 3: Figure A2. ATP changes during trophozoite development. A tightly synchronized culture of Plasmodium falciparum 3D7 parasites was obtained by enriching trophozoite/schizont-infected red blood cells by centrifugation through 60% Percoll, incubating the enriched cells with fresh red blood cells in culture medium for 8 hours, followed by sorbitol treatment. After a further overnight incubation, the parasites had reached the trophozoite stage (0 hours image above) and ATP levels were measured every 2 hours over an 8 hour period. Representative images of Giemsa-stained thin-smears of parasitized red blood cells at the various time points of trophozoite development used in the ATP time-course assay are shown below the graph. (PDF 580 KB)
12936_2012_2509_MOESM3_ESM.pdf
Authors’ original file for figure 1
12936_2012_2509_MOESM4_ESM.pdf
Authors’ original file for figure 2
12936_2012_2509_MOESM5_ESM.png
Authors’ original file for figure 3
12936_2012_2509_MOESM6_ESM.png
Authors’ original file for figure 4
12936_2012_2509_MOESM7_ESM.png
Authors’ original file for figure 5
12936_2012_2509_MOESM8_ESM.png
Literatur
Über diesen Artikel

Weitere Artikel der Ausgabe 1/2012

Malaria Journal 1/2012 Zur Ausgabe

Neu im Fachgebiet Innere Medizin

Meistgelesene Bücher aus der Inneren Medizin

2017 | Buch

Rheumatologie aus der Praxis

Entzündliche Gelenkerkrankungen – mit Fallbeispielen

Dieses Fachbuch macht mit den wichtigsten chronisch entzündlichen Gelenk- und Wirbelsäulenerkrankungen vertraut. Anhand von über 40 instruktiven Fallbeispielen werden anschaulich diagnostisches Vorgehen, therapeutisches Ansprechen und der Verlauf …

Herausgeber:
Rudolf Puchner

2016 | Buch

Ambulant erworbene Pneumonie

Was, wann, warum – Dieses Buch bietet differenzierte Diagnostik und Therapie der ambulant erworbenen Pneumonie zur sofortigen sicheren Anwendung. Entsprechend der neuesten Studien und Leitlinien aller wichtigen Fachgesellschaften.

Herausgeber:
Santiago Ewig

Mail Icon II Newsletter

Bestellen Sie unseren kostenlosen Newsletter Update Innere Medizin und bleiben Sie gut informiert – ganz bequem per eMail.

© Springer Medizin 

Bildnachweise