Ring stage dormancy of Plasmodium falciparum tolerant to artemisinin and its analogues - A genetically regulated "Sleeping Beauty".

The appearance in 2008 in western Cambodia of Plasmodium falciparum tolerant to artemisinin, defined by longer parasite clearance time following drug administration and in vitro by a slightly higher survival rate of the ring stage after a 3-h treatment with 700 nM artemisinin (or analogues, collectively termed ART), has raised concerns of the possible loss of this frontline antimalarial [used in the form of an artemisinin combination therapy (ACT)], with its low IC50 value against the ring stage and pleiotropic pro-drug/poison property. The key genetic marker of ART tolerance phenotype is a number of non-synonymous mutations in Pfkelch13 propeller domain. This results in defective assembly at the ring stage of a cytostome structure located at cytoplasmic side of the parasite membrane required for invagination of a double-membrane endosome carrying host cytosol haemoglobin to the digestive vacuole. The consequential deprivation of amino acids initiates ring stage parasites bearing the causal mutations in PfK13 (or other key cytostome components) entry into a dormant state ("Sleeping Beauty"), which, after a duration longer than that the short-lived ART, "Sleeping Beauty" ring parasite resumes its normal, but accelerated, development to maintain the 48-h intra-erythrocytic life-cycle. We posit that when ART-tolerant P. falciparum has acquired under ART stress the causative PfK13 mutation (not obligatory if mutations occur in other critical cytostome components), together with other necessary mutations to adjust to the new normalcy and to provide survival competitiveness, ART-tolerant parasite has now evolved into a genetically programmed "Sleeping Beauty". The onus of preventing the spread of ART-tolerant P. falciparum lies with the efficacy of ACT partner drug, hence the recommendation of a triple ACT (TACT). Nevertheless, attention should also be focussed on understanding the mechanisms of dormancy, such as induction, maintenance and recovery, to enable discovery and development of novel antimalarials targeting this unique parasite stage.

Lumefantrine attenuates Plasmodium falciparum artemisinin resistance during the early ring stage.

Emerging artemisinin resistance in Plasmodium falciparum malaria has the potential to become a global public health crisis. In Southeast Asia, this phenomenon clinically manifests in the form of delayed parasite clearance following artemisinin treatment. Reduced artemisinin susceptibility is limited to the early ring stage window, which is sufficient to allow parasites to survive the short half-life of artemisinin exposure. A screen of known clinically-implemented antimalarial drugs was performed to identify a drug capable of enhancing the killing activity of artemisinins during this critical resistance window. As a result, lumefantrine was found to increase the killing activity of artemisinin against an artemisinin-resistant clinical isolate harboring the C580Y kelch13 mutation. Isobologram analysis revealed synergism during the early ring stage resistance window, when lumefantrine was combined with artemether, an artemisinin derivative clinically partnered with lumefantrine. These findings suggest that lumefantrine should be clinically explored as a partner drug in artemisinin-based combination therapies to control emerging artemisinin resistance.

Universal scanning-free initiation of eukaryote protein translation-a new normal.

A unique feature of eukaryote initiation of protein translation is a so-called scanning of 5'-untranslated region (5'-UTR) by a ribosome initiation complex to enable bound Met-tRNAi access to the initiation codon located further downstream. Here, we propose a universal scanning-free translation initiation model that is independent of 5'-UTR length and applicable to both 5'-m7G (capped) and uncapped mRNAs.

Plug for the parasitophorous duct: a solution of two conundra.

BACKGROUND: We present two conundra in the biology of intraerythrocytic malaria parasite: how an apparent open parasitophorous duct provide direct access of only a select set of serum proteins to the parasitophorous vacuole, and how proteases mediate membrane lysis to allow merozoite egress. SOLUTION: We posit the existence of a parasitophorous vacuolar duct plug that is originally formed from a tight junction (or parts thereof) between merozoite apical surface and red blood cell plasma membrane, which by moving over the parasite surface towards the posterior end draws the parasite into the host cell interior, and by remaining at the passage orifice provides a location of transporter(s) for import of serum proteins into parasitophorous vacuole and an opening for merozoite egress upon its dissolution/dismantling through protease(s) action. CONCLUSION: This notion obviates the need of a distinct intact parasitophorous vacuolar membrane, which in the proposed model is an extension of the red blood cell membrane but still forms an intracellular compartment for parasite growth and development. The model is testable using existing high-resolution electron and X-ray tomography tools.

Overexpression of plasmepsin II and plasmepsin III does not directly cause reduction in Plasmodium falciparum sensitivity to artesunate, chloroquine and piperaquine.

Artemisinin derivatives and their partner drugs in artemisinin combination therapies (ACTs) have played a pivotal role in global malaria mortality reduction during the last two decades. The loss of artemisinin efficacy due to evolving drug-resistant parasites could become a serious global health threat. Dihydroartemisinin-piperaquine is a well tolerated and generally highly effective ACT. The implementation of a partner drug in ACTs is critical in the control of emerging artemisinin resistance. Even though artemisinin is highly effective in parasite clearance, it is labile in the human body. A partner drug is necessary for killing the remaining parasites when the pulses of artemisinin have ceased. A population of Plasmodium falciparum parasites in Cambodia and adjacent countries has become resistant to piperaquine. Increased copy number of the genes encoding the haemoglobinases Plasmepsin II and Plasmepsin III has been linked with piperaquine resistance by genome-wide association studies and in clinical trials, leading to the use of increased plasmepsin II/plasmepsin III copy number as a molecular marker for piperaquine resistance. Here we demonstrate that overexpression of plasmepsin II and plasmepsin III in the 3D7 genetic background failed to change the susceptibility of P. falciparum to artemisinin, chloroquine and piperaquine by both a standard dose-response analysis and a piperaquine survival assay. Whilst plasmepsin copy number polymorphism is currently implemented as a molecular surveillance resistance marker, further studies to discover the molecular basis of piperaquine resistance and potential epistatic interactions are needed.

Fitness Loss under Amino Acid Starvation in Artemisinin-Resistant Plasmodium falciparum Isolates from Cambodia.

Artemisinin is the most rapidly effective drug for Plasmodium falciparum malaria treatment currently in clinical use. Emerging artemisinin-resistant parasites pose a great global health risk. At present, the level of artemisinin resistance is still relatively low with evidence pointing towards a trade-off between artemisinin resistance and fitness loss. Here we show that artemisinin-resistant P. falciparum isolates from Cambodia manifested fitness loss, showing fewer progenies during the intra-erythrocytic developmental cycle. The loss in fitness was exacerbated under the condition of low exogenous amino acid supply. The resistant parasites failed to undergo maturation, whereas their drug-sensitive counterparts were able to complete the erythrocytic cycle under conditions of amino acid deprivation. The artemisinin-resistant phenotype was not stable, and loss of the phenotype was associated with changes in the expression of a putative target, Exp1, a membrane glutathione transferase. Analysis of SNPs in haemoglobin processing genes revealed associations with parasite clearance times, suggesting changes in haemoglobin catabolism may contribute to artemisinin resistance. These findings on fitness and protein homeostasis could provide clues on how to contain emerging artemisinin-resistant parasites.

Comparative genome analysis between Southeast Asian and South American Zika viruses.

OBJECTIVE: To understand the cause for the differences between potentially mild Southeast Asian and the more pathogenic ZIKV in South America. METHODS: A comparative genomic analysis was performed to determine putative causations stemming from ZIKV. RESULTS: Phylogenetic analyses integrating geographical and time factors revealed that Southeast Asian ZIKV might not be the direct source of South American outbreaks as previously speculated. Amino acid residues unique to South American ZIKV isolates at the envelope, pr and NS1 proteins are listed and shown in the structural context. These unique residues on external viral proteins are not found in Southeast Asian ZIKV and could be responsible for the ongoing outbreak either via an intrinsic property of the virus or interactions with human immunity. Only a selected few primer/probe sets currently in clinical use were identified of being capable of detecting ZIKV strains worldwide. The envelope proteins of dengue virus (DENV) and ZIKV also showed a remarkable degree of similarity especially at the surface residues. CONCLUSIONS: These findings may help explain the cross-reactivity of DENV antibodies to ZIKV. Thus, major caveats must be exercised in using existing diagnostic tools for ZIKV.

Plasmodium falciparum malaria: Convergent evolutionary trajectories towards delayed clearance following artemisinin treatment.

Malaria is a major global health challenge with 300million new cases every year. The most effective regimen for treating Plasmodium falciparum malaria is based on artemisinin and its derivatives. The drugs are highly effective, resulting in rapid clearance of parasites even in severe P. falciparum malaria patients. During the last five years, artemisinin-resistant parasites have begun to emerge first in Cambodia and now in Thailand and Myanmar. At present, the level of artemisinin resistance is relatively low with clinical presentation of delayed artemisinin clearance (a longer time to reduce parasite load) and a small decrease in artemisinin sensitivity in cultured isolates. Nevertheless, multiple genetic loci associated with delayed parasite clearance have been reported, but they cannot account for a large portion of cases. Even the most well-studied kelch 13 propeller mutations cannot always predict the outcome of artemisinin treatment in vitro and in vivo. Here we propose that delayed clearance by artemisinin could be the result of convergent evolution, driven by multiple trajectories to overcome artemisinin-induced stress, but precluded to become full blown resistance by high fitness cost. Genetic association studies by several genome-wide approaches reveal linkage disequilibrium between multiple loci and delayed parasite clearance. Genetic manipulations at some of these loci already have resulted in loss in artemisinin sensitivity. The notion presented here is by itself consistent with existing evidence on artemisinin resistance and has the potential to be explored using available genomic data. Most important of all, molecular surveillance of artemisinin resistance based on multi-genic markers could be more informative than relying on any one particular molecular marker.

Comparative genome-wide analysis and evolutionary history of haemoglobin-processing and haem detoxification enzymes in malarial parasites.

BACKGROUND: Malaria parasites have evolved a series of intricate mechanisms to survive and propagate within host red blood cells. Intra-erythrocytic parasitism requires these organisms to digest haemoglobin and detoxify iron-bound haem. These tasks are executed by haemoglobin-specific proteases and haem biocrystallization factors that are components of a large multi-subunit complex. Since haemoglobin processing machineries are functionally and genetically linked to the modes of action and resistance mechanisms of several anti-malarial drugs, an understanding of their evolutionary history is important for drug development and drug resistance prevention. METHODS: Maximum likelihood trees of genetic repertoires encoding haemoglobin processing machineries within Plasmodium species, and with the representatives of Apicomplexan species with various host tropisms, were created. Genetic variants were mapped onto existing three-dimensional structures. Genome-wide single nucleotide polymorphism data were used to analyse the selective pressure and the effect of these mutations at the structural level. RESULTS: Recent expansions in the falcipain and plasmepsin repertoires are unique to human malaria parasites especially in the Plasmodium falciparum and P. reichenowi lineage. Expansion of haemoglobin-specific plasmepsins occurred after the separation event of Plasmodium species, but the other members of the plasmepsin family were evolutionarily conserved with one copy for each sub-group in every Apicomplexan species. Haemoglobin-specific falcipains are separated from invasion-related falcipain, and their expansions within one specific locus arose independently in both P. falciparum and P. vivax lineages. Gene conversion between P. falciparum falcipain 2A and 2B was observed in artemisinin-resistant strains. Comparison between the numbers of non-synonymous and synonymous mutations suggests a strong selective pressure at falcipain and plasmepsin genes. The locations of amino acid changes from non-synonymous mutations mapped onto protein structures revealed clusters of amino acid residues in close proximity or near the active sites of proteases. CONCLUSION: A high degree of polymorphism at the haemoglobin processing genes implicates an imposition of selective pressure. The identification in recent years of functional redundancy of haemoglobin-specific proteases makes them less appealing as potential drug targets, but their expansions, especially in the human malaria parasite lineages, unequivocally point toward their functional significance during the independent and repetitive adaptation events in malaria parasite evolutionary history.

Synergism of antimalarial antibiotics with hydrogen peroxide in inhibiting Plasmodium falciparum growth in culture.

Although morbidity and mortality from malaria have steadily decreased worldwide, the ever present menace of the appearance of Plasmodium falciparum resistant to all antimalarials in current use, including most recently to artemisinin and its analogs, is of utmost concern, especially when development of new and affordable antimalarials has not kept abreast of this phenomenon. An alternative approach is to identify synergistic drug combinations, which would allow employment of otherwise non-efficacious antimalarial drugs. This study demonstrates that combinations of the chemical oxidant hydrogen hydroxide with antimalarial antibiotics targeting parasite mitochondrial and apicoplast ribosomes, which normally produce 'delayed-death' of parasites, act synergistically to inhibit P. falciparum growth in culture.

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.

Role of altholactone in inducing type II apoptosis signalling pathway and expression of cancer-related genes in cervical carcinoma HeLa cell line.

Goniothalamus species (Annonaceae) is a shrub that grows in the rainforest of tropical Asia. Several compounds have been isolated and exhibit the potential use for cancer treatment. In this work, altholactone isolated from Goniothalamus macrophyllus was investigated for its cytotoxicity, apoptosis signalling and the expression of cancer-related genes in the cervical carcinoma HeLa cells. Cytotoxicity was evaluated by MTT assay. Apoptotic characteristics were evaluated by morphological studies. Caspase-3 activity was detected using a fluorogenic substrate. Cytochrome c release from mitochondria and protein Bid were determined by Western blotting and cancer-related genes expression by RT-PCR. The results demonstrated that altholactone was cytotoxic to HeLa (IC50 = 9.6 µg/mL), and apoptotic cell death was manifested by appearance of chromatin condensation and caspase-3 activation, which was inhibited by specific inhibitors of both caspase-8 and -9. Release into the cytosol of cytochrome and cleavage of Bid occurred. Altholactone also caused a decrease in bcl-2 and an increase in p53 expression. These unique properties of altholactone suggest a potential for cancer chemotherapy.

Plasma membrane Ca(2+)-ATPase sulfhydryl modifications: implication for oxidized red cell.

A common perturbation found in cells under oxidative stress is alteration in cellular Ca2+ homeostasis. In order to understand the effects of such oxidative damage, human red cell plasma membrane Ca(2+)-ATPase (PMCA) was studied by measuring PMCA activity, both in the presence and absence of calmodulin (CaM), following treatment with sulfhydryl agents, N-ethylmaleimide, iodoacetate and diamide. PMCA activity of washed red cell membrane was measured by coupling with pyruvate kinase, using phosphoenolpyruvate as substrate, and lactate dehydrogenase to convert pyruvate to lactate employing beta-NADH as co-factor. All treatments inhibited basal and CaM-stimulated activity in a dose-dependent manner (0.01-1 mM), but at low concentrations, basal Ca(2+)-ATPase activity was inhibited whereas CaM-stimulated activity was unaffected. Inhibition by diamide, a disulfide-forming agent, was reversed with dithiotreitol (DTT). Treatment with calpain, a calcium-dependent protease, elevated basal PMCA activity to CaM-stimulated level, but abolished response to CaM. Further treatment with diamide inhibited PMCA activity, which could be restored by DTT, but only to basal and not CaM-stimulated level. These studies indicated that it is necessary to protect against both sulfhydryl and proteolytic damages to red cell PMCA if perturbation to Ca2+ homeostasis is to be minimized. This has implications for membranes under oxidative stress, such as in the hereditary anemia, thalassemia, where membrane-bound unmatched hemoglobin chains cause oxidative damage to red blood cells.

Alpha-hemoglobin stabilizing protein: molecular function and clinical correlation.

The discovery of alpha-hemoglobin stabilizing protein (AHSP), a chaperone for free alpha-hemoglobin (alpha-Hb), has provided a satisfactory solution to the perplexing problem of balanced globin levels for Hb production in erythroid cells in the face of a two-fold excess of alpha-globin to beta-globin gene dosage. Unmatched alpha-Hb is unstable and precipitates onto membranes, where the released heme exerts oxidative damages resulting in ineffective erythropoiesis and hemolytic anemia, the underlying causes of pathology in the hereditary anemia of beta-thalassemia. The interaction of alpha-Hb with AHSP involves surfaces normally employed in binding to beta-Hb. However, a conformational change to the AHSP-bound alpha-Hb results in an oxidized heme, but in a pocket that is now less exposed to the outside environment, thereby protecting against both peroxide-induced heme loss and iron-induced redox reaction. Studies in both mice and humans indicate that reduction in AHSP can result in hematological pathology. Conversely, alpha-Hb variants that are compromised in their ability to bind with AHSP produce beta-thalassemia-like symptoms. Disease conditions like some forms of thalassemia that are directly associated with AHSP structural and/or functional defects can now be included within the category of chaperonopathies.

Mechanisms of the action of povidone-iodine against human and avian influenza A viruses: its effects on hemagglutination and sialidase activities.

BACKGROUND: Influenza virus infection causes significant morbidity and mortality and has marked social and economic impacts throughout the world. The influenza surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), act cooperatively to support efficient influenza A virus replication and provide the most important targets for anti-influenza chemotherapy. In this study, povidone-iodine (PVP-I), which has a broad-spectrum microbicidal property, was examined for its inhibitory effects against influenza virus infection in MDCK cells and the mechanisms of PVP-I action on HA and NA were revealed. RESULTS: Results obtained using a novel fluorescence- and chromogenic-based plaque inhibition assay showed that 1.56 mg/ml PVP-I inhibited infections in MDCK cells of human (8 strains) and avian (5 strains) influenza A viruses, including H1N1, H3N2, H5N3 and H9N2, from 23.0-97.5%. A sialidase inhibition assay revealed that PVP-I inhibited N1, N2 and N3 neuraminidases with IC50 values of 9.5-212.1 microg/ml by a mixed-type inhibition mechanism. Receptor binding inhibition and hemagglutinin inhibition assays indicated that PVP-I affected viral hemagglutinin rather than host-specific sialic acid receptors. CONCLUSION: Mechanisms of reduction of viral growth in MDCK cells by PVP-I involve blockade of viral attachment to cellular receptors and inhibition of viral release and spread from infected cells. Therefore, PVP-I is useful to prevent infection and limit spread of human and avian influenza viruses.

Simple, efficient, and cost-effective multiplex genotyping with matrix assisted laser desorption/ionization time-of-flight mass spectrometry of hemoglobin beta gene mutations.

A number of common mutations in the hemoglobin beta (HBB) gene cause beta-thalassemia, a monogenic disease with high prevalence in certain ethnic groups. As there are 30 HBB variants that cover more than 99.5% of HBB mutant alleles in the Thai population, an efficient and cost-effective screening method is required. Three panels of multiplex primer extensions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were developed. The first panel simultaneously detected 21 of the most common HBB mutations, while the second panel screened nine additional mutations, plus seven of the first panel for confirmation; the third panel was used to confirm three HBB mutations, yielding a 9-Da mass difference that could not be clearly distinguished by the previous two panels. The protocol was both standardized using 40 samples of known genotypes and subsequently validated in 162 blind samples with 27 different genotypes (including a normal control), comprising heterozygous, compound heterozygous, and homozygous beta-thalassemia. Results were in complete agreement with those from the genotyping results, conducted using three different methods overall. The method developed here permitted the detection of mutations missed using a single genotyping procedure. The procedure should serve as the method of choice for HBB genotyping due to its accuracy, sensitivity, and cost-effectiveness, and can be applied to studies of other gene variants that are potential disease biomarkers.

Analysis of N-glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses.

The initial step essential in influenza virus infection is specific binding of viral hemagglutinin to host cell-surface glycan receptors. Influenza A virus specificity for the host is mediated by viral envelope hemagglutinin, that binds to receptors containing glycans with terminal sialic acids. Human viruses preferentially bind to alpha2-->6 linked sialic acids on receptors of host cells, whereas avian viruses are specific for the alpha2-->3 linkage on the target cells. Human influenza virus isolates more efficiently infect amniotic membrane (AM) cells than chorioallantoic membrane (CAM) cells. N-glycans were isolated from AM and CAM cells of 10-day-old chicken embryonated eggs and their structures were analyzed by multi-dimensional HPLC mapping and MALDI-TOF-MS techniques. Terminal N-acetylneuraminic acid contents in the two cell types were similar. However, molar percents of alpha2-->3 linkage preferentially bound by avian influenza virus were 27.2 in CAM cells and 15.4 in AM cells, whereas those of alpha2-->6 linkage favored by human influenza virus were 8.3 (CAM) and 14.2 (AM). Molar percents of sulfated glycans, recognized by human influenza virus, in CAM and AM cells were 3.8 and 12.7, respectively. These results have revealed structures and molar percents of N-glycans in CAM and AM cells important in determining human and avian influenza virus infection and viral adaptation.

A genetically hard-wired metabolic transcriptome in Plasmodium falciparum fails to mount protective responses to lethal antifolates.

Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites.

Inhibition of alpha-globin gene expression by RNAi.

RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 5' region of exon II of alpha-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1x10(6) ECFCs were transfected with lipofectamine-containing alpha-globin specific siRNA. After 48h of transfection, alpha-globin specific siRNA produced significantly reduction of alpha-globin mRNA level in a dose-dependent manner, but it did not affect the level of beta-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this alpha-globin mRNA specific siRNA.