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The characterization of de novo mutations in regions of high sequence and structural diversity from whole-genome sequencing data remains highly challenging. Complex structural variants tend to arise in regions of high repetitiveness and low complexity, challenging both de novo assembly, in which short reads do not capture the long-range context required for resolution, and mapping approaches, in which improper alignment of reads to a reference genome that is highly diverged from that of the sample can lead to false or partial calls. Long-read technologies can potentially solve such problems but are currently unfeasible to use at scale. Here we present Corticall, a graph-based method that combines the advantages of multiple technologies and prior data sources to detect arbitrary classes of genetic variant. We construct multisample, colored de Bruijn graphs from short-read data for all samples, align long-read–derived haplotypes and multiple reference data sources to restore graph connectivity information, and call variants using graph path-finding algorithms and a model for simultaneous alignment and recombination. We validate and evaluate the approach using extensive simulations and use it to characterize the rate and spectrum of de novo mutation events in 119 progeny from four Plasmodium falciparumexperimental crosses, using long-read data on the parents to inform reconstructions of the progeny and to detect several known and novel nonallelic homologous recombination events.

Drug-resistant micro-organisms became widespread in the 20th Century, often with devastating consequences, in response to widespread use of natural and synthetic drugs against infectious diseases. Antimalarial resistance provides one of the earliest examples, following the introduction of new medicines that filled important needs for prophylaxis and treatment around the globe. In the present chapter, we offer a brief synopsis of major antimalarial developments from two natural remedies, the qinghaosu and cinchona bark infusions, and of synthetic drugs inspired by the active components of these remedies. We review some contributions that early efficacy studies of antimalarial treatment brought to clinical pharmacology, including convincing documentation of atebrine-resistant malaria in the 1940s, prior to the launching of what soon became first-choice antimalarials, chloroquine and amodiaquine. Finally, we discuss some new observations on the molecular genetics of drug resistance, including delayed parasite clearances that have been increasingly observed in response to artemisinin derivatives in regions of South-East Asia.

Plasmodium falciparummalaria parasites carry ∼60 vargenes that encode variable adhesins termed P. falciparumerythrocyte membrane protein-1. Clonal expression of a single P. falciparumerythrocyte membrane protein-1 variant on the surface of the parasitized host erythrocyte promotes binding of the cell to blood elements (including noninfected erythrocytes, leukocytes) and walls of microvessels. These binding events enable parasitized erythrocytes to sequester and avoid clearance by the spleen, and they also contribute to disease by causing microvascular inflammation and obstruction.

Although selection of hemoglobin C (HbC) by malaria has been speculated for decades, only recently have epidemiologic studies provided support for HbC protection against malaria in West Africa. A reduced risk of malaria associated with the homozygous CC state has been attributed to the inability of CC cells to support parasite multiplication in vitro. However, there have been conflicting data and conclusions regarding the ability of CC red cells to support parasite replication. Reports that parasites cannot multiply in CC cells in vitro contrast with detection of substantial parasite densities in CC patients with malaria. We have therefore investigated Plasmodium falciparum growth in CC cells in vitro. Our data show that the multiplication rate of several P falciparum lines is measurable in CC cells, but lower than that in AA (HbA-normal) cells. A high proportion of ring forms and trophozoites disintegrates within a subset of CC cells, an observation that accounts for the overall lower replication rate. In addition, knobs present on the surface of infected CC cells are fewer in number and morphologically aberrant when compared with those on AA cells. Events in malaria pathogenesis that involve remodeling of the erythrocyte surface and the display of parasite antigens may be affected by these knob abnormalities. Our data suggest that only a subset of CC cells supports normal parasite replication and that components of malaria protection associated with the CC state may affect the parasite's replication capacity and involve aberrant knob formation on CC cells.

Although selection of hemoglobin C (HbC) by malaria has been speculated for decades, only recently have epidemiologic studies provided support for HbC protection against malaria in West Africa. A reduced risk of malaria associated with the homozygous CC state has been attributed to the inability of CC cells to support parasite multiplication in vitro. However, there have been conflicting data and conclusions regarding the ability of CC red cells to support parasite replication. Reports that parasites cannot multiply in CC cells in vitro contrast with detection of substantial parasite densities in CC patients with malaria. We have therefore investigatedPlasmodium falciparumgrowth in CC cells in vitro. Our data show that the multiplication rate of severalP falciparumlines is measurable in CC cells, but lower than that in AA (HbA-normal) cells. A high proportion of ring forms and trophozoites disintegrates within a subset of CC cells, an observation that accounts for the overall lower replication rate. In addition, knobs present on the surface of infected CC cells are fewer in number and morphologically aberrant when compared with those on AA cells. Events in malaria pathogenesis that involve remodeling of the erythrocyte surface and the display of parasite antigens may be affected by these knob abnormalities. Our data suggest that only a subset of CC cells supports normal parasite replication and that components of malaria protection associated with the CC state may affect the parasite’s replication capacity and involve aberrant knob formation on CC cells.

The malaria hypothesis proposes a survival advantage for individuals with hemoglobin variants in areas of endemicPlasmodium falciparum malaria. Hemoglobin C (HbC) is a possible example in West Africa, where this hemoglobin has a centric distribution with high frequencies among certain populations including the Dogon ethnic group. To test whether HbC is associated with protection from malaria, we performed a case-control study in the Dogon of Bandiagara, Mali. HbC was present in 68 of 391 (17.4%) of uncomplicated malaria control cases, whereas it was detected in only 3 of 67 cases (4.5%) of severe malaria (odds ratio [OR], 0.22;P?=?.01). Further, HbC was present in only 1 of 34 cases (2.9%) with cerebral manifestations, the most common presentation of severe malaria in this population (OR, 0.14; P?=?.03). Episodes of uncomplicated malaria and parasitemias (4800-205?050/µL) were identified in cases of homozygous HbC (HbCC), which indicates thatP falciparum parasites are able to efficiently replicate within HbCC erythrocytes in vivo. These findings suggest that HbC does not protect against infection or uncomplicated malaria but can protect against severe malaria in the Dogon population of Bandiagara, Mali. The data also suggest that the protective effect associated with HbC may be greater than that of HbS in this population.

The major first-line treatment for malaria, especially the deadliest form caused by Plasmodium falciparum, is combination therapy with an artemisinin-based drug (ART) plus a partner drug to assure complete cure. Without an effective partner drug, ART administration alone can fail because of the ability of small populations of blood-stage malaria parasites to enter into a dormant state and survive repeated treatments for a week or more.

Genome analysis of the Plasmodium falciparummalaria parasite already is identifying genes relevant to therapeutic- and vaccine-related research. The genetic blueprint of P. falciparumwill ultimately need to be understood at multiple levels of an integrated system and will provide a detailed account of the life processes of the parasite and of the devastating disease it causes.

A gene (pfhsp86) encoding a member of the heat-shock protein 90 (HSP90) family has been isolated from Plasmodium falciparum(Pf). The pfhsp86coding region comprises two exons separated by an 0.8-kb intron with consensus splice junction sequences. The transcript itself is 3.4-kb long and includes a 0.65-kb 5'-untranslated region (UTR) and a 0.54-kb 3'-UTR. Upstream of the transcription start point (tsp) are putative promoter modules: an inverted CCAAT box, a G + C-rich sequence and several TATA sequences. Transcription is enhanced in erythrocyte-stage parasites cultivated at elevated temperatures (2–3-fold at 39°C and 3-4-fold at 41 °C, relative to 37°C). The pfhsp86gene maps within a chromosome 7 segment that is linked to chloroquine (Cq) response in a Pfcross. The parents of this cross (Dd2, HB3) differ in the first exon by two trinucleotide repeats, while more divergence is apparent between the introns. These trinucleotide repeat differences are linked to Cq response in the HB3 × Dd2 cross, but they did not predict Cq response in nine Pflines from different locations.

A total of 507 simple sequence repeats (SSRs or “microsatellites”) were identified fromPlasmodium falciparumsequences in GenBank and from inserts in a genomic DNA library. Oligonucleotide primers from sequences that flank 224 of these SSRs were synthesized and used in PCR assays to test for simple sequence length polymorphisms (SSLPs). Of the 224 SSRs, 188 showed SSLPs among 12 differentP. falciparumlines; 116 of these SSLPs were assigned to chromosome linkage groups by physical mapping and by comparing their inheritance patterns against those of restriction fragment length polymorphism markers in a genetic cross (HB3×Dd2). The predominant SSLPs inP. falciparumwere found to contain [TA]n, [T]n, and [TAA]n, a feature that is reminiscent of plant genomes and is consistent with the proposed algal-like origin of malaria parasites. Since such SSLPs are abundant and readily isolated, they are a powerful resource for genetic analysis ofP. falciparum.

The antigenic variation and cytoadherence of Plasmodium falciparum-infected erythrocytes are modulated by a family of variant surface proteins encoded by the varmultigene family. The vargenes occur on multiple chromosomes, often in clusters, and 50 to 150 genes are estimated to be present in the haploid parasite genome. Transcripts from vargenes have been previously mapped to internal chromosome positions, but the generality of such assignments and the expression sites and mechanisms that control switches of vargene expression are still in early stages of investigation. Here we describe investigations of closely related vargenes that occur in association with repetitive elements near the telomeres of P. falciparumchromosomes. DNA sequence analysis of one of these genes (FCR3-varT11-1) shows the characteristic two-exon structure encoding expected varfeatures, including three variable Duffy binding-like (DBL) domains, a transmembrane sequence, and a carboxy-terminal segment thought to anchor the protein product in knobs at the surface of the parasitized erythrocyte. FCR3-varT11-1cross-hybridizes with vargenes located close to the telomeres of many other P. falciparumchromosomes, including a transcribed gene (FCR3-varT3-1) in chromosome 3 of the P. falciparumFCR3 line. The relatively high level transcription from this gene shows that the polymorphic chromosome ends of P. falciparum, which have been proposed to be transcriptionally silent, can be active expression sites for vargenes. The pattern of the FCR3-varT11-1and FCR3-varT3-1genes are variable between different P. falciparumlines, presumably due to DNA rearrangements. Thus, recombination events in subtelomeric DNA may have a role in the expression of novel varforms.

The malaria organism Plasmodium falciparumdetoxifies heme released during degradation of host erythrocyte hemoglobin by sequestering it within the parasite digestive vacuole as a polymer called hemozoin. Antimalarial agents such as chloroquine appear to work by interrupting the heme polymerization process, but their efficacy has been impaired by the emergence of drug-resistant organisms. We report here the identification of a new class of antimalarial compounds, hexadentate ethylenediamine-N,N′-bis[propyl(2-hydroxy-(R)-benzylimino)]metal(III) complexes [(R)-ENBPI-M(III)] and a corresponding ((R)-benzylamino)] analog [(R)-ENBPA-M(III)], a group of lipophilic monocationic leads amenable to metallopharmaceutical development. Racemic mixtures of Al(III), Fe(III), or Ga(III) but not In(III) (R)-ENBPI metallo-complexes killed intraerythrocytic malaria parasites in a stage-specific manner, the R= 4,6-dimethoxy-substituted ENBPI Fe(III) complex being most potent (IC50∼1 μM). Inhibiting both chloroquine-sensitive and -resistant parasites, potency of these imino complexes correlated in a free metal-independent manner with their ability to inhibit heme polymerization in vitroIn contrast, the reduced (amino) 3-MeO-ENBPA Ga(III) complex (MR045) was found to be selectively toxic to chloroquine-resistant parasites in a verapamil-insensitive manner. In 21 independent recombinant progeny of a genetic cross, susceptibility to this agent mapped in perfect linkage with the chloroquine resistance phenotype suggesting that a locus for 3-MeO-ENBPA Ga(III) susceptibility was located on the same 36-kilobase segment of chromosome 7 as the chloroquine resistance determinant. These compounds may be useful as novel probes of chloroquine resistance mechanisms and for antimalarial drug development.

Su, X.-Z., Carucci, D. J., and Wellems, T. E. 1998.Plasmodium falciparum:Parasite typing by using a multicopy microsatellite marker, PfRRM.Experimental Parasitology89,262–265.

Aptamer-based fluorescent sensors specifically detect the antimalarial drugs piperaquine and mefloquine from patient samples and crushed tablets.

WR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodiumtransfectants, selectively targets the parasite’s dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing the human dhfrgene have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2).

Introns of Plasmodium falciparum var genes act as transcriptional silencing elements that help control antigenic variations. In transfected episomes, intron silencing of a drug-selectable marker under var promoter control is reversed by the spontaneous deletion of key intron regions. The resulting promoter activation does not affect the transcription of chromosomal var genes.

ABSTRACTIntrons of Plasmodium falciparum vargenes act as transcriptional silencing elements that help control antigenic variations. In transfected episomes, intron silencing of a drug-selectable marker under varpromoter control is reversed by the spontaneous deletion of key intron regions. The resulting promoter activation does not affect the transcription of chromosomal vargenes.

Su, X., and Wellems, T. E. 1999.Plasmodium falciparum;Assignment of microsatellite markers to chromosomes by PFG–PCR.Experimental Parasitology91, 367–369.

Protozoan Plasmodiumparasites are the causative agents of malaria, a deadly disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild or severe symptoms, or fatal, depending on many factors such as parasite virulence and host immune status. Malaria can be treated with various drugs, with artemisinin-based combination therapies (ACTs) being the first-line choice.

ABSTRACTPlasmodium vivaxcauses heavy burdens of disease across malarious regions worldwide. Mature P. vivaxasexual and transmissive gametocyte stages occur in the blood circulation, and it is often assumed that accumulation/sequestration in tissues is not an important phase in their development. Here, we present a systematic study of P. vivaxstage distributions in infected tissues of nonhuman primate (NHP) malaria models as well as in blood from human infections. In a comparative analysis of the transcriptomes of P. vivaxand Plasmodium falciparumblood-stage parasites, we found a conserved cascade of stage-specific gene expression despite the greatly different gametocyte maturity times of these two species. Using this knowledge, we validated a set of conserved asexual- and gametocyte-stage markers both by quantitative real-time PCR and by antibody assays of peripheral blood samples from infected patients and NHP (Aotussp.). Histological analyses of P. vivaxparasites in organs of 13 infected NHP (Aotusand Saimirispecies) demonstrated a major fraction of immature gametocytes in the parenchyma of the bone marrow, while asexual schizont forms were enriched to a somewhat lesser extent in this region of the bone marrow as well as in sinusoids of the liver. These findings suggest that the bone marrow is an important reservoir for gametocyte development and proliferation of malaria parasites.IMPORTANCEPlasmodium vivaxmalaria continues to cause major public health burdens worldwide. Yet, significant knowledge gaps in the basic biology and epidemiology of P. vivaxmalaria remain, largely due to limited available tools for research and diagnostics. Here, we present a systematic examination of tissue sequestration during P. vivaxinfection. Studies of nonhuman primates and malaria patients revealed enrichment of developing sexual stages (gametocytes) and mature replicative stages (schizonts) in the bone marrow and liver, relative to those present in peripheral blood. Identification of the bone marrow as a major P. vivaxtissue reservoir has important implications for parasite diagnosis and treatment.

ABSTRACTSome Kelch mutations of the Plasmodium falciparumK13 protein confer increased survival to dihydroartemisinin (DHA)-treated ring-stage parasites. Here, we asked if K13 mutations affect a dormancy phenotype allowing parasites to survive DHA exposure and then sorbitol selection. Although recrudescence from dormancy differed between two distinct parasite lines, it was similar for isogenic lines carrying single-site substitutions in K13. Therefore, K13 mutations do not alter the DHA-sorbitol combined dormancy phenotype; rather, traits from other loci likely determine this phenotype.

Background: The human malaria parasite Plasmodium falciparumsurvives pressures from the host immune system and antimalarial drugs by modifying its genome. Genetic recombination and nucleotide substitution are the two major mechanisms that the parasite employs to generate genome diversity. A better understanding of these mechanisms may provide important information for studying parasite evolution, immune evasion and drug resistance. Results: Here, we used a high-density tiling array to estimate the genetic recombination rate among 32 progeny of a P. falciparumgenetic cross (7G8 × GB4). We detected 638 recombination events and constructed a high-resolution genetic map. Comparing genetic and physical maps, we obtained an overall recombination rate of 9.6 kb per centimorgan and identified 54 candidate recombination hotspots. Similar to centromeres in other organisms, the sequences of P. falciparumcentromeres are found in chromosome regions largely devoid of recombination activity. Motifs enriched in hotspots were also identified, including a 12-bp G/C-rich motif with 3-bp periodicity that may interact with a protein containing 11 predicted zinc finger arrays. Conclusions: These results show that the P. falciparumgenome has a high recombination rate, although it also follows the overall rule of meiosis in eukaryotes with an average of approximately one crossover per chromosome per meiosis. GC-rich repetitive motifs identified in the hotspot sequences may play a role in the high recombination rate observed. The lack of recombination activity in centromeric regions is consistent with the observations of reduced recombination near the centromeres of other organisms.

Size variations in homologous chromosomes from six Giardia lamblia isolates have been demonstrated. Four or five intensely stained (major) bands as well as a variable number of lightly stained (minor) bands are present in pulsed field gradient separations. Southern blot analysis with total chromosomal DNA as well as chromosome specific probes indicates that each minor band cross-hybridizes with a major band. Minor bands of doubly cloned organisms appear identical to those of parent clones, indicating that the minor bands do not reflect the presence of variant members within the total population of trophozoites. Densitometric comparisons of chromosome bands from known numbers of Plasmodium falciparum ring stage forms and known numbers of Giardia trophozoites suggest that minor bands MBa and MBb are present in each Giardia trophozoite. Comparison of Not I restriction fragments from the major and minor bands reveals common restriction fragments. Taken together, the data imply that sets of closely related chromosomes occur in the Giardia trophozoite.

A procedure for in situ hybridization to polytene chromosomes from the ovarian nurse cells of Anopheles gambiae Giles has been developed. This procedure involves a modification of established methods for Drosophila larval salivary gland polytene chromosomes. Treatment of chromosome squashes with xylene followed by slow rehydration provides required resolution of chromosome banding patterns, possibly because fatty contaminants are removed from ovarian nurse cell preparations. Using this procedure, unique DNA sequences from a genomic library of An. gambiae have been mapped on adult mosquito polytene chromosomes. The ability to locate genetic markers on chromosomes will allow correlation of physical and genetic maps. Such maps will facilitate identification of genetic loci and expand our knowledge of the genomic organization of An. gambiae.