Abstract
BackgroundMalaria, a major public health issue in developing nations, is responsible for more than one million deaths a year. The most lethal species, Plasmodium falciparum, causes up to 90% of fatalities. Drug resistant strains to common therapies have emerged worldwide and recent artemisinin-based combination therapy failures hasten the need for new antimalarial drugs. Discovering novel compounds to be used as antimalarials is expedited by the use of a high-throughput screen (HTS) to detect parasite growth and proliferation. Fluorescent dyes that bind to DNA have replaced expensive traditional radioisotope incorporation for HTS growth assays, but do not give additional information regarding the parasite stage affected by the drug and a better indication of the drug's mode of action. Live cell imaging with RNA dyes, which correlates with cell growth and proliferation, has been limited by the availability of successful commercial dyes.ResultsAfter screening a library of newly synthesized stryrl dyes, we discovered three RNA binding dyes that provide morphological details of live parasites. Utilizing an inverted confocal imaging platform, live cell imaging of parasites increases parasite detection, improves the spatial and temporal resolution of the parasite under drug treatments, and can resolve morphological changes in individual cells.ConclusionThis simple one-step technique is suitable for automation in a microplate format for novel antimalarial compound HTS. We have developed a new P. falciparum RNA high-content imaging growth inhibition assay that is robust with time and energy efficiency.
Highlights
Malaria, a major public health issue in developing nations, is responsible for more than one million deaths a year
Live cell imaging was performed using the Pathway HT microscope with temperature regulated at 37°C
Our Pathway HT is customized with high efficiency Semrock bandpass filters, and samples were imaged with all fluorescence combinations of Semrock DAPI, GFP, FRET, CFP, YFP, and Texas Red BrightLine filter sets to measure fluorescence intensity of the initial 125 RNA probes screened
Summary
A major public health issue in developing nations, is responsible for more than one million deaths a year. Drug resistant strains to common therapies have emerged worldwide and recent artemisinin-based combination therapy failures hasten the need for new antimalarial drugs. Malaria continues to be a major public health issue in many parts of the developing world [1]. The emergence of chloroquine-resistant parasites and DDT-resistant mosquito vectors has led to a reappearance and spread of malaria in most of the developing world. With the absence of an efficient vaccine, worldwide resistance to all commonly used antimalarial drugs (quinine, aminoquinolines and antifolate derivates), and the concern of emerging resistance to our last defense against this disease (artemisinin-based combination therapies) [2], there is a dire need for new antimalarial strategies
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