Quantitative Dissection of Clone-Specific Growth Rates in Cultured Malaria Parasites

Measurement of parasite proliferation in cultured red blood cells underpins many facets of malaria research, from drug sensitivity assays to assessing the impact of experimentally altered genes on parasite growth, virulence, and fitness. Pioneering efforts to grow Plasmodium falciparum in cultured red blood cells revolutionized malaria research and spurred the development of semi-high throughput growth assays using radio-labeled hypoxanthine, an essential nucleic acid precursor, as a reporter of whole-cycle proliferation (Trager and Jensen, 1976; Desjardins et al., 1979). Use of hypoxanthine (Hx) and other surrogate readouts of whole-cycle proliferation remains the dominant choice in malaria research. While amenable to high-throughput inference of bulk proliferation rates, these assays are blind to the underlying developmental and cellular steps of growth in human red blood cells. Modern whole-genome methods promise to reveal much about basic parasite biology, but progress is hindered by limitations of our ability to precisely quantify the specific development and growth events within the erythrocytic cycle. Here we build on standard visual and Hx-incorporation measures of growth by quantifying sub-phenotypes of a rapid proliferator, the multi-drug resistant clone Dd2, from a standard wild type clone, HB3. These data illustrate differences in cycle duration, merozoite production, and invasion rate and efficiency that underpin Dd2’s average 2-fold proliferation advantage over HB3 per erythrocytic cycle. The ability to measure refined growth phenotypes can inform the development of high-throughput methods to isolate molecular and developmental determinants of differential parasite growth rates

An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins

Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.This work was funded by grants from the National Institutes of Health to BS (AI 64671) and funds provided by the University of California, Riverside to KLR. SA was supported by a predoctoral fellowship from the American Heart Association, and GGD by a C.J. Martin Overseas Fellowship from the Australian National Health and Medical Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies

Antimalarial chemotherapy, globally reliant on artemisinin-based combination therapies (ACTs), is threatened by the spread of drug resistance in Plasmodium falciparum parasites. Here we use zinc-finger nucleases to genetically modify the multidrug resistance-1 transporter PfMDR1 at amino acids 86 and 184, and demonstrate that the widely prevalent N86Y mutation augments resistance to the ACT partner drug amodiaquine and the former first-line agent chloroquine. In contrast, N86Y increases parasite susceptibility to the partner drugs lumefantrine and mefloquine, and the active artemisinin metabolite dihydroartemisinin. The PfMDR1 N86 plus Y184F isoform moderately reduces piperaquine potency in strains expressing an Asian/African variant of the chloroquine resistance transporter PfCRT. Mutations in both digestive vacuole-resident transporters are thought to differentially regulate ACT drug interactions with host haem, a product of parasite-mediated haemoglobin degradation. Global mapping of these mutations illustrates where the different ACTs could be selectively deployed to optimize treatment based on regional differences in PfMDR1 haplotypes.This work was funded in part by the National Institutes of Health (R01 AI50234, AI124678 and AI109023) and a Burroughs Wellcome Fund Investigator in Pathogenesis of Infectious Diseases award to D.A.F. This research also received funding from the Portuguese Fundacao para a Ciencia e Tecnologia (FCT), cofunded by Programa Operacional Regional do Norte (ON.2-O Novo Norte); from the Quadro de Referencia Estrategico Nacional (QREN) through the Fundo Europeu de Desenvolvimento Regional (FEDER) and from the Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). M.I.V. is the recipient of a postdoctoral fellowship from FCT/Ministerio da Ciencia e Ensino Superior, Portugal-MCES (SFRH/BPD/76614/2011). A.M.L. was supported by an Australian National Health and Medical Research Council (NHMRC) Overseas Biomedical Fellowship (585519). R.E.M. was supported by an NHMRC RD Wright Biomedical Fellowship (1053082). A.C.U. was supported by an Irving scholarship from Columbia University. We thank Dr Andrea Ecker for her help with plasmid design and Pedro Ferreira for his expert help with Fig. 6.info:eu-repo/semantics/publishedVersio

Drug Resistance in Eukaryotic Microorganisms

Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies

Quinine Treatment Selects the pfnhe-1 ms4760-1 Polymorphism in Malian Patients with Falciparum Malaria

Background. The mechanism of Plasmodium falciparum resistance to quinine is not known. In vitro quantitative trait loci mapping suggests involvement of a predicted P. falciparum sodium-hydrogen exchanger (pfnhe-1) on chromosome 13. Methods. We conducted prospective quinine efficacy studies in 2 villages, Kolle and Faladie, Mali. Cases of clinical malaria requiring intravenous therapy were treated with standard doses of quinine and followed for 28 days. Treatment outcomes were classified using modified World Health Organization protocols. Molecular markers of parasite polymorphisms were used to distinguish recrudescent parasites from new infections. The prevalence of pfnhe-1 ms4760-1 among parasites before versus after quinine treatment was determined by direct sequencing. Results. Overall, 163 patients were enrolled and successfully followed. Without molecular correction, the mean adequate clinical and parasitological response (ACPR) was 50.3% (n = 163). After polymerase chain reaction correction to account for new infections, the corrected ACPR was 100%. The prevalence of ms4760-1 increased significantly, from 26.2% (n = 107) before quinine treatment to 46.3% (n = 54) after therapy (P = .01). In a control sulfadoxine-pyrimethamine study, the prevalence of ms4760-1 was similar before and after treatment. Conclusions. This study supports a role for pfnhe-1 in decreased susceptibility of P. falciparum to quinine in the field.Howard Hughes Medical Institute [55005502]; Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health; European and Developing Countries Clinical Trials Partnership [EDCTP IP_07_31060_002]info:eu-repo/semantics/publishedVersio

Hackathons as a means of accelerating scientific discoveries and knowledge transfer

International audienceScientific research plays a key role in the advancement of human knowledge and pursuit of solutions to important societal challenges. Typically, research occurs within specific institutions where data are generated and subsequently analyzed. Although collaborative science bringing together multiple institutions is now common, in such collaborations the analytical processing of the data is often performed by individual researchers within the team, with only limited internal oversight and critical analysis of the workflow prior to publication. Here, we show how hackathons can be a means of enhancing collab-orative science by enabling peer review before results of analyses are published by cross-validating the design of studies or underlying data sets and by driving reproducibility of scientific analyses. Traditionally, in data analysis processes, data generators and bioinformaticians are divided and do not collaborate on analyzing the data. Hackathons are a good strategy to build bridges over the traditional divide and are potentially a great agile extension to the more structured collaborations between multiple investigators and institutions

In-Home Training for Fathers of Children with Autism: A Follow up Study and Evaluation of Four Individual Training Components

Literature regarding fathers of children with autism remains sparse, and because mothers are the more common intervening parent, few training methods have focused on fathers. Thus, we sought to evaluate effects of in-home training directed at fathers and their ability to train mothers in the same manner in which they were trained. Fathers were taught four skills commonly associated with in-home training interventions for parents of children with autism: following the child’s lead, imitation with animation, commenting on the child, and expectant waiting. Father skills were evaluated twice a week for 12 weeks during videotaped in-home father–child play sessions. Analyses included visual inspection of graphed data and statistical analyses of father skill acquisition, mother skill acquisition, and child behaviors with both parents. A multivariate repeated measures analysis of 18 dyads revealed significant increases in frequencies of fathers’ imitation with animation, expectant waiting, and commenting on the child. Child initiating rates increased significantly as did frequencies of child non-speech vocalizations. Analysis of mothers revealed significant increases in frequencies of imitation with animation, expectant waiting, and following the child’s lead. Child behaviors had similar results for father and mother sessions. Findings are consistent with those from our first study indicating that fathers can effectively implement skills that promote father–child social interactions and that children respond positively to this approach

Measuring growth, resistance, and recovery after artemisinin treatment of Plasmodium falciparum in a single semi-high-throughput assay

Background: Artemisinin partial resistance (ART-R) has spread throughout Southeast Asia and mutations in Pfkelch13, the molecular marker of resistance, are widely reported in East Africa. Effective in vitro assays and robust phenotypes are crucial for monitoring populations for the emergence and spread of resistance. The recently developed extended Recovery Ring-stage Survival Assay used a qPCR-based readout to reduce the labour intensiveness for in vitro phenotyping of ART-R and improved correlation with the clinical phenotype of ART-R. Here, the assay is extended and refined to include measurements of parasite growth and recovery after drug exposure. Clinical isolates and progeny from two genetic crosses were used to optimize and validate the reliability of a straight-from-blood, SYBR Green-based qPCR protocol in a 96-well plate format to accurately measure phenotypes with this new Growth, Resistance, and Recovery assay (GRRA). Results: The assay determined growth between 6 and 96 h, resistance at 120 h, and recovery from 120 to 192 h. Growth can be accurately captured by qPCR and is shown by reproduction of previous growth phenotypes from HB3 × Dd2. Resistance measured at 120 h continually shows the most consistent phenotype for ring stage susceptibility. Recovery identifies an additional response to drug in parasites that are determined sensitive by replicative viability at 120 h. Comparison of progeny phenotypes for Growth versus Resistance showed a minor but significant correlation, whereas Growth versus Recovery and Resistance versus Recovery showed no significant correlation. Additionally, dried blood spot (DBS) samples matched replicative viability measured from liquid samples demonstrating Resistance can be easily quantified using either storage method. Conclusions: The direct-from-blood qPCR-based methodology provides the throughput needed to quickly measure large numbers of parasites for multiple relevant phenotypes. Growth can reveal fitness defects and illuminate relationships between proliferation rates and drug response. Recovery serves as a complementary phenotype to resistance that quantifies the ability of sensitive parasites to tolerate drug exposure. All three phenotypes offer a comprehensive assessment of parasite-drug interaction each with potential independent genetic determinants of main effect and overlapping secondary effects. By adapting the method to include DBS, readouts can be easily extended to ex vivo surveillance applications

Hackathons as a means of accelerating scientific discoveries and knowledge transfer

Scientific research plays a key role in the advancement of human knowledge and pursuit of solutions to important societal challenges. Typically, research occurs within specific institutions where data are generated and subsequently analyzed. Although collaborative science bringing together multiple institutions is now common, in such collaborations the analytical processing of the data is often performed by individual researchers within the team, with only limited internal oversight and critical analysis of the workflow prior to publication. Here, we show how hackathons can be a means of enhancing collaborative science by enabling peer review before results of analyses are published by cross-validating the design of studies or underlying data sets and by driving reproducibility of scientific analyses. Traditionally, in data analysis processes, data generators and bioinformaticians are divided and do not collaborate on analyzing the data. Hackathons are a good strategy to build bridges over the traditional divide and are potentially a great agile extension to the more structured collaborations between multiple investigators and institutions