Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model.

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.

The 2 micron plasmid purloins the yeast cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation?

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes.

Plasmodium vivax chloroquine resistance links to pvcrt transcription in a genetic cross.

Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.

A novel role for the mitotic spindle during DNA segregation in yeast: promoting 2 microm plasmid-cohesin association.

The 2 microm circle plasmid in Saccharomyces cerevisiae is a model for a stable, high-copy-number, extrachromosomal "selfish" DNA element. By combining a partitioning system and an amplification system, the plasmid ensures its stable propagation and copy number maintenance, even though it does not provide any selective advantage to its host. Recent evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation. We now demonstrate an unexpected and unconventional role for the mitotic spindle in the plasmid-partitioning pathway. The spindle specifies the nuclear address of the 2 microm circle and promotes recruitment of the cohesin complex to the plasmid-partitioning locus STB. Only the nuclear microtubules, and not the cytoplasmic ones, are required for loading cohesin at STB. In cells recovering from nocodazole-induced spindle depolymerization and G(2)/M arrest, cohesin-STB association can be established coincident with spindle restoration. This postreplication recruitment of cohesin is not functional in equipartitioning. However, normally acquired cohesin can be inactivated after replication without causing plasmid missegregation. In the mtw1-1 mutant yeast strain, the plasmid cosegregates with the spindle and the spindle-associated chromosomes; by contrast, a substantial number of the chromosomes are not associated with the spindle. These results are consistent with a model in which the spindle promotes plasmid segregation in a chromosome-linked fashion.

The 2 microm plasmid causes cell death in Saccharomyces cerevisiae with a mutation in Ulp1 protease.

The 2 microm circle plasmid confers no phenotype in wild-type Saccharomyces cerevisiae but in a nib1 mutant, an elevated plasmid copy number is associated with cell death. Complementation was used to identify nib1 as a mutant allele of the ULP1 gene that encodes a protease required for removal of a ubiquitin-like protein, Smt3/SUMO, from protein substrates. The nib1 mutation replaces conserved tryptophan 490 with leucine in the protease domain of Ulp1. Complete deletion of ULP1 is lethal, even in a strain that lacks the 2 microm circle. Partial deletion of ULP1, like the nib1 mutation, results in clonal variations in plasmid copy number. In addition, a subset of these mutant cells produces lineages in which all cells have reduced proliferative capacity, and this phenotype is dependent upon the presence of the 2 microm circle. Segregation of the 2 microm circle requires two plasmid-encoded proteins, Rep1 and Rep2, which were found to colocalize with Ulp1 protein in the nucleus and interact with Smt3 in a two-hybrid assay. These associations and the observation of missegregation of a fluorescently tagged 2 microm circle reporter plasmid in a subset of ulp1 mutant cells suggest that Smt3 modification plays a role in both plasmid copy number control and segregation.

Mutations in a partitioning protein and altered chromatin structure at the partitioning locus prevent cohesin recruitment by the Saccharomyces cerevisiae plasmid and cause plasmid missegregation.

The 2 microm circle is a highly persistent "selfish" DNA element resident in the Saccharomyces cerevisiae nucleus whose stability approaches that of the chromosomes. The plasmid partitioning system, consisting of two plasmid-encoded proteins, Rep1p and Rep2p, and a cis-acting locus, STB, apparently feeds into the chromosome segregation pathway. The Rep proteins assist the recruitment of the yeast cohesin complex to STB during the S phase, presumably to apportion the replicated plasmid molecules equally to daughter cells. The DNA-protein and protein-protein interactions of the partitioning system, as well as the chromatin organization at STB, are important for cohesin recruitment. Rep1p variants that are incompetent in binding to Rep2p, STB, or both fail to assist the assembly of the cohesin complex at STB and are nonfunctional in plasmid maintenance. Preventing the cohesin-STB association without impeding Rep1p-Rep2p-STB interactions also causes plasmid missegregation. During the yeast cell cycle, the Rep1p and Rep2p proteins are expelled from STB during a short interval between the late G(1) and early S phases. This dissociation and reassociation event ensures that cohesin loading at STB is replication dependent and is coordinated with chromosomal cohesin recruitment. In an rsc2 Delta yeast strain lacking a specific chromatin remodeling complex and exhibiting a high degree of plasmid loss, neither Rep1p nor the cohesin complex can be recruited to STB. The phenotypes of the Rep1p mutations and of the rsc2 Delta mutant are consistent with the role of cohesin in plasmid partitioning being analogous to that in chromosome partitioning.

Stable propagation of 'selfish' genetic elements.

Extrachromosomal or chromosomally integrated genetic elements are common among prokaryotic and eukaryotic cells. These elements exhibit a variety of 'selfish' strategies to ensure their replication and propagation during the growth of their host cells. To establish long-term persistence, they have to moderate the degree of selfishness so as not to imperil the fitness of their hosts. Earlier genetic and biochemical studies together with more recent cell biological investigations have revealed details of the partitioning mechanisms employed by low copy bacterial plasmids. At least some bacterial chromosomes also appear to rely on similar mechanisms for their own segregation. The 2 mm plasmid of Saccharomyces cerevisiae and related yeast plasmids provide models for optimized eukaryotic selfish DNA elements. Selfish DNA elements exploit the genetic endowments of their hosts without imposing an undue metabolic burden on them. The partitioning systems of these plasmids appear to make use of a molecular trick by which the plasmids feed into the segregation pathway established for the host chromosomes.

A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax.

The Plasmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable, and reproducible in vitro liver-stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum, including the release of infected merozoites and infection of overlaid erythrocytes, as well as the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput antimalarial drug screening and vaccine development.

Controlled human malaria infections by intradermal injection of cryopreserved Plasmodium falciparum sporozoites.

Controlled human malaria infection with sporozoites is a standardized and powerful tool for evaluation of malaria vaccine and drug efficacy but so far only applied by exposure to bites of Plasmodium falciparum (Pf)-infected mosquitoes. We assessed in an open label Phase 1 trial, infection after intradermal injection of respectively 2,500, 10,000, or 25,000 aseptic, purified, vialed, cryopreserved Pf sporozoites (PfSPZ) in three groups (N = 6/group) of healthy Dutch volunteers. Infection was safe and parasitemia developed in 15 of 18 volunteers (84%), 5 of 6 volunteers in each group. There were no differences between groups in time until parasitemia by microscopy or quantitative polymerase chain reaction, parasite kinetics, clinical symptoms, or laboratory values. This is the first successful infection by needle and syringe with PfSPZ manufactured in compliance with regulatory standards. After further optimization, the use of such PfSPZ may facilitate and accelerate clinical development of novel malaria drugs and vaccines.

Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine.

Consistent, high-level, vaccine-induced protection against human malaria has only been achieved by inoculation of Plasmodium falciparum (Pf) sporozoites (SPZ) by mosquito bites. We report that the PfSPZ Vaccine--composed of attenuated, aseptic, purified, cryopreserved PfSPZ--was safe and well tolerated when administered four to six times intravenously (IV) to 40 adults. Zero of six subjects receiving five doses and three of nine subjects receiving four doses of 1.35 × 10(5) PfSPZ Vaccine and five of six nonvaccinated controls developed malaria after controlled human malaria infection (P = 0.015 in the five-dose group and P = 0.028 for overall, both versus controls). PfSPZ-specific antibody and T cell responses were dose-dependent. These data indicate that there is a dose-dependent immunological threshold for establishing high-level protection against malaria that can be achieved with IV administration of a vaccine that is safe and meets regulatory standards.

Female Anopheles gambiae antennae: increased transcript accumulation of the mosquito-specific odorant-binding-protein OBP2.

BACKGROUND: New interventions are required to optimally and sustainably control the Anopheles sp. mosquitoes that transmit malaria and filariasis. The mosquito olfactory system is important in host seeking (transmission) and mate finding (reproduction). Understanding olfactory function could lead to development of control strategies based on repelling parasite-carrying mosquitoes or attracting them into a fatal trap. FINDINGS: Our initial focus is on odorant binding proteins with differential transcript accumulation between female and male mosquitoes. We report that the odorant binding protein, OBP2 (AGAP003306), had increased expression in the antennae of female vs. male Anopheles gambiae sensu stricto (G3 strain). The increased expression in antennae of females of this gene by quantitative RT-PCR was 4.2 to 32.3 fold in three independent biological replicates and two technical replicate experiments using A. gambiae from two different laboratories. OBP2 is a member of the vast OBP superfamily of insect odorant binding proteins and belongs to the predominantly dipteran clade that includes the Culex oviposition kairomone-binding OBP1. Phylogenetic analysis indicates that its orthologs are present across culicid mosquitoes and are likely to play a conserved role in recognizing a molecule that might be critical for female behavior. CONCLUSIONS: OBP2 has increased mRNA transcript accumulation in the antennae of female as compared to male A. gambiae. This molecule and related molecules may play an important role in female mosquito feeding and breeding behavior. This finding may be a step toward providing a foundation for understanding mosquito olfactory requirements and developing control strategies based on reducing mosquito feeding and breeding success.

Establishment of an in vitro assay for assessing the effects of drugs on the liver stages of Plasmodium vivax malaria.

Plasmodium vivax (Pv) is the second most important human malaria parasite. Recent data indicate that the impact of Pv malaria on the health and economies of the developing world has been dramatically underestimated. Pv has a unique feature in its life cycle. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain as dormant forms called hypnozoites, which cause relapses of malaria months to several years after the primary infection. Elimination of malaria caused by Pv will be facilitated by developing a safe, highly effective drug that eliminates Pv liver stages, including hypnozoites. Identification and development of such a drug would be facilitated by the development of a medium to high throughput assay for screening drugs against Pv liver stages. We undertook the present pilot study to (1) assess the feasibility of producing large quantities of purified, vialed, cryopreserved Pv sporozoites and (2) establish a system for culturing the liver stages of Pv in order to assess the effects of drugs on the liver stages of Pv. We used primaquine (PQ) to establish this assay model, because PQ is the only licensed drug known to clear all Pv hepatocyte stages, including hypnozoites, and the effect of PQ on Pv hepatocyte stage development in vitro has not previously been reported. We report that we have established the capacity to reproducibly infect hepatoma cells with purified, cyropreserved Pv spz from the same lot, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. These data provide the foundation for finalizing a medium throughput, high content assay to identify new drugs for the elimination of all Pv liver stages.

Evolutionarily conserved multisubunit RBL2/p130 and E2F4 protein complex represses human cell cycle-dependent genes in quiescence.

The mammalian Retinoblastoma (RB) family including pRB, p107, and p130 represses E2F target genes through mechanisms that are not fully understood. In D. melanogaster, RB-dependent repression is mediated in part by the multisubunit protein complex Drosophila RBF, E2F, and Myb (dREAM) that contains homologs of the C. elegans synthetic multivulva class B (synMuvB) gene products. Using an integrated approach combining proteomics, genomics, and bioinformatic analyses, we identified a p130 complex termed DP, RB-like, E2F, and MuvB (DREAM) that contains mammalian homologs of synMuvB proteins LIN-9, LIN-37, LIN-52, LIN-54, and LIN-53/RBBP4. DREAM bound to more than 800 human promoters in G0 and was required for repression of E2F target genes. In S phase, MuvB proteins dissociated from p130 and formed a distinct submodule that bound MYB. This work reveals an evolutionarily conserved multisubunit protein complex that contains p130 and E2F4, but not pRB, and mediates the repression of cell cycle-dependent genes in quiescence.

Further evidence for BRCA1 communication with the inactive X chromosome.

BRCA1, a breast and ovarian cancer-suppressor gene, exerts tumor-suppressing functions that appear to be associated, at least in part, with its DNA repair, checkpoint, and mitotic regulatory activities. Earlier work from our laboratory also suggested an ability of BRCA1 to communicate with the inactive X chromosome (Xi) in female somatic cells (Ganesan et al., 2002). Xiao et al. (2007) (this issue of Cell) have challenged this conclusion. Here we discuss recently published data from our laboratory and others and present new results that, together, provide further support for a role of BRCA1 in the regulation of XIST concentration on Xi in somatic cells.

Segregation of the yeast plasmid: similarities and contrasts with bacterial plasmid partitioning.

The high copy yeast plasmid 2 microm circle, like the well-studied low copy bacterial plasmids, utilizes two partitioning proteins and a cis-acting 'centromere'-like sequence for its stable propagation. Functionally, though, the protein and DNA constituents of the two partitioning systems are quite distinct. Key events in the yeast and bacterial segregation pathways are plasmid organization, localization, replication, 'counting' of replicated molecules and their distribution to daughter cells. We suggest that the two systems facilitate these common logistical steps by adapting to the physical, biochemical, and mechanical contexts in which the host chromosomes segregate.