Plasmodium parasites mount an arrest response to dihydroartemisinin, as revealed by whole transcriptome shotgun sequencing (RNA-seq) and microarray study.

BACKGROUND: Control of malaria is threatened by emerging parasite resistance to artemisinin and derivative drug (ART) therapies. The molecular detail of how Plasmodium malaria parasites respond to ART and how this could contribute to resistance are not well understood. To address this question, we performed a transcriptomic study of dihydroartemisinin (DHA) response in P. falciparum K1 strain and in P. berghei ANKA strain using microarray and RNA-seq technology. RESULTS: Microarray data from DHA-treated P. falciparum trophozoite stage parasites revealed a response pattern that is overall less trophozoite-like and more like the other stages of asexual development. A meta-analysis of these data with previously published data from other ART treatments revealed a set of common differentially expressed genes. Notably, ribosomal protein genes are down-regulated in response to ART. A similar pattern of trophozoite transcriptomic change was observed from RNA-seq data. RNA-seq data from DHA-treated P. falciparum rings reveal a more muted response, although there is considerable overlap of differentially expressed genes with DHA-treated trophozoites. No genes are differentially expressed in DHA-treated P. falciparum schizonts. The transcriptional response of P. berghei to DHA treatment in vivo in infected mice is similar to the P. falciparum in vitro culture ring and trophozoite responses, in which ribosomal protein genes are notably down-regulated. CONCLUSIONS: Ring and trophozoite stage Plasmodium respond to ART by arresting metabolic processes such as protein synthesis and glycolysis. This response can be protective in rings, as shown by the phenomenon of dormancy. In contrast, this response is not as protective in trophozoites owing to their commitment to a highly active and vulnerable metabolic state. The lower metabolic demands of schizonts could explain why they are less sensitive and unresponsive to ART. The ART response pattern is revealed clearly from RNA-seq data, suggesting that this technology is of great utility for studying drug response in Plasmodium.

Origin of robustness in generating drug-resistant malaria parasites.

Biological robustness allows mutations to accumulate while maintaining functional phenotypes. Despite its crucial role in evolutionary processes, the mechanistic details of how robustness originates remain elusive. Using an evolutionary trajectory analysis approach, we demonstrate how robustness evolved in malaria parasites under selective pressure from an antimalarial drug inhibiting the folate synthesis pathway. A series of four nonsynonymous amino acid substitutions at the targeted enzyme, dihydrofolate reductase (DHFR), render the parasites highly resistant to the antifolate drug pyrimethamine. Nevertheless, the stepwise gain of these four dhfr mutations results in tradeoffs between pyrimethamine resistance and parasite fitness. Here, we report the epistatic interaction between dhfr mutations and amplification of the gene encoding the first upstream enzyme in the folate pathway, GTP cyclohydrolase I (GCH1). gch1 amplification confers low level pyrimethamine resistance and would thus be selected for by pyrimethamine treatment. Interestingly, the gch1 amplification can then be co-opted by the parasites because it reduces the cost of acquiring drug-resistant dhfr mutations downstream in the same metabolic pathway. The compensation of compromised fitness by extra GCH1 is an example of how robustness can evolve in a system and thus expand the accessibility of evolutionary trajectories leading toward highly resistant alleles. The evolution of robustness during the gain of drug-resistant mutations has broad implications for both the development of new drugs and molecular surveillance for resistance to existing drugs.

An automatic device for detection and classification of malaria parasite species in thick blood film.

BACKGROUND: Current malaria diagnosis relies primarily on microscopic examination of Giemsa-stained thick and thin blood films. This method requires vigorously trained technicians to efficiently detect and classify the malaria parasite species such as Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) for an appropriate drug administration. However, accurate classification of parasite species is difficult to achieve because of inherent technical limitations and human inconsistency. To improve performance of malaria parasite classification, many researchers have proposed automated malaria detection devices using digital image analysis. These image processing tools, however, focus on detection of parasites on thin blood films, which may not detect the existence of parasites due to the parasite scarcity on the thin blood film. The problem is aggravated with low parasitemia condition. Automated detection and classification of parasites on thick blood films, which contain more numbers of parasite per detection area, would address the previous limitation. RESULTS: The prototype of an automatic malaria parasite identification system is equipped with mountable motorized units for controlling the movements of objective lens and microscope stage. This unit was tested for its precision to move objective lens (vertical movement, z-axis) and microscope stage (in x- and y-horizontal movements). The average precision of x-, y- and z-axes movements were 71.481 ± 7.266 µm, 40.009 ± 0.000 µm, and 7.540 ± 0.889 nm, respectively. Classification of parasites on 60 Giemsa-stained thick blood films (40 blood films containing infected red blood cells and 20 control blood films of normal red blood cells) was tested using the image analysis module. By comparing our results with the ones verified by trained malaria microscopists, the prototype detected parasite-positive and parasite-negative blood films at the rate of 95% and 68.5% accuracy, respectively. For classification performance, the thick blood films with Pv parasite was correctly classified with the success rate of 75% while the accuracy of Pf classification was 90%. CONCLUSIONS: This work presents an automatic device for both detection and classification of malaria parasite species on thick blood film. The system is based on digital image analysis and featured with motorized stage units, designed to easily be mounted on most conventional light microscopes used in the endemic areas. The constructed motorized module could control the movements of objective lens and microscope stage at high precision for effective acquisition of quality images for analysis. The analysis program could accurately classify parasite species, into Pf or Pv, based on distribution of chromatin size.

Enhancement of DNA hybridization under acoustic streaming with three-piezoelectric-transducer system.

Recently, we have demonstrated that DNA hybridization using acoustic streaming induced by two piezoelectric transducers provides higher DNA hybridization efficiency than the conventional method. In this work, we refine acoustic streaming system for DNA hybridization by inserting an additional piezoelectric transducer and redesigning the locations of the transducers. The Comsol® Multiphysics was used to design and simulate the velocity field generated by the piezoelectric agitation. The simulated velocity vector followed a spiral vortex flow field with an average direction outward from the center of the transducers. These vortices caused the lower signal intensity in the middle of the microarray for the two-piezoelectric disk design. On the contrary, the problem almost disappeared in the three-piezoelectric-disk system. The optimum condition for controlling the piezoelectric was obtained from the dye experiments with different activation settings for the transducers. The best setting was to activate the side disks and middle disk alternatively with 1 second activating time and 3 second non-activating time for both sets of transducers. DNA hybridization using microarrays for the malaria parasite Plasmodium falciparum from the optimized process yielded a three-fold enhancement of the signal compared to the conventional method. Moreover, a greater number of spots passed quality control in the optimized device, which could greatly improve biological interpretation of DNA hybridization data.

Global gene expression profiling of Plasmodium falciparum in response to the anti-malarial drug pyronaridine.

BACKGROUND: Pyronaridine (PN) and chloroquine (CQ) are structurally related anti-malarial drugs with primarily the same mode of action. However, PN is effective against several multidrug-resistant lines of Plasmodium falciparum, including CQ resistant lines, suggestive of important operational differences between the two drugs. METHODS: Synchronized trophozoite stage cultures of P. falciparum strain K1 (CQ resistant) were exposed to 50% inhibitory concentrations (IC50) of PN and CQ, and parasites were harvested from culture after 4 and 24 hours exposure. Global transcriptional changes effected by drug treatment were investigated using DNA microarrays. RESULTS: After a 4 h drug exposure, PN induced a greater degree of transcriptional perturbation (61 differentially expressed features) than CQ (10 features). More genes were found to respond to 24 h treatments with both drugs, and 461 features were found to be significantly responsive to one or both drugs across all treatment conditions. Filtering was employed to remove features unrelated to primary drug action, specifically features representing genes developmentally regulated, secondary stress/death related processes and sexual stage development. The only significant gene ontologies represented among the 46 remaining features after filtering relate to host exported proteins from multi-gene families. CONCLUSIONS: The malaria parasite's molecular responses to PN and CQ treatment are similar in terms of the genes and pathways affected. However, PN appears to exert a more rapid response than CQ. The faster action of PN may explain why PN is more efficacious than CQ, particularly against CQ resistant isolates. In agreement with several other microarray studies of drug action on the parasite, it is not possible, however, to discern mechanism of drug action from the drug-responsive genes.

DNA hybridization enhancement using piezoelectric microagitation through a liquid coupling medium.

In conventional DNA microarray hybridization, delivery of target cDNAs to surface-bounded probes depends solely on diffusion, which is notoriously slow, and thus typically requires 6-20 h to complete. In this study, piezoelectric microagitation through a liquid coupling medium is employed to enhance DNA hybridization efficiency and the results are compared with the standard static hybridization method. DNA hybridization was performed in a sealed aluminium chamber containing DNA microarray glass chip, coupling medium and piezoelectric transducers. 3×SSC (Saline Sodium Citrate) was used as a coupling medium to prevent overheating of the piezoelectric transducers and to effectively transmit ultrasonic wave to the glass chip. Flow visualization using fluidic dye and velocimetry (PTV) technique was applied to observe fluid transport in the hybridization chamber. It was revealed that the dye solution was homogeneously distributed within 10 min under dynamic agitation while it took over 1 h to reach the same level of homogeneity in static condition. Plasmodium falciparum DNA microarrays and total RNA extracted from parasite cells were used as a model for DNA microarray experiments. It was found that the required hybridization time may be substantially reduced from 16 h to 4 h by the use of dynamic hybridization scheme. With the same hybridization time of 16 h, dynamic hybridization resulted in higher fluorescent signals of ∼33% and ∼24% compared to static hybridization in Cy3 and Cy5 channels, respectively. Additionally, good/effective spots, some of which were not formed by static method, were enhanced and distributed more uniformly over the microarray. Therefore, the developed dynamic hybridization with integrated piezoelectric microagitation platform is highly promising for DNA analysis in molecular biology and medical applications.