Alison M. Creasey

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites

Artemisinin‐ and artesunate‐resistant Plasmodium chabaudi mutants, AS‐ART and AS‐ATN, were previously selected from chloroquine‐resistant clones AS‐30CQ and AS‐15CQ respectively. Now, a genetic cross between AS‐ART and the artemisinin‐sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS‐30CQ and AS‐ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS‐ATN to artemisinin derivates, the other cannot account solely for the resistance of AS‐ART, relative to the responses of its sensitive progenitor AS‐30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug‐response phenotype was not genetically stable. No mutations in the UBP‐1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.

A marked seasonality of malaria transmsission in two rural sites in eastern Sudan

The ecology of Anopheles arabiensis and its relationship to malaria transmission was investigated in two villages in eastern Sudan. Seasonal malaria case incidence was compared with the number of vectors detected and with climatic variables. Following the end of the short rainy season in October the number of A. arabiensis detected dropped gradually until February when neither outdoor human bait trapping nor indoor spray catches revealed any mosquitoes. Vectors re-appeared in June as humidity rose with the onset of rain. Despite the apparent absence of the vector at the height of the long, hot dry season between February and May, sporadic asymptomatic malaria infections were detected in the two villages. The low endemicity of malaria in the area was reflected by the relatively low total September-December parasite and sporozoite rates (15 and 1.4%, respectively) measured in the villages. The entomological inoculation rate (EIR) was estimated to be around two to three infective bites per person per year, although heterogeneity in the transmission indices of malaria between the two villages was observed. The implications of these patterns of anopheline population dynamics for the epidemiology and control of malaria in eastern Sudan are considered.

Genetic diversity of Plasmodium falciparum in a village in eastern Sudan. 1. Diversity of enzymes, 2D-PAGE proteins and antigens

Twenty-nine plasmodium falciparum isolates from patients in Asar village, eastern Sudan, were characterized for variation in 18 different genetically controlled characters, including iso-enzymes, proteins detected by two-dimensional polyacrylamide gel electrophoresis and blood-stage antigens. Considerable allelic diversity in the genes determining these characters was detected. Each isolate contained genetically distinct parasites. Fifteen individuals were infected with more than one parasite genotype. The diversity of parasite types is most probably generated by recombination during mosquito transmission of mixed parasite clones.

Plasmodium falciparum: An abundant stage-specific protein expressed during early gametocyte development

A stage-specific protein has been identified in gametocytes of Plasmodium falciparum. The protein is represented on two-dimensional electrophoresis by peptides of two apparent Mr of 27,000 and 25,000, each of which has at least four different isoelectric points between pH 6.0 and 5.0. The protein is designated Pfg 27/25 (P. falciparum gametocyte-specific antigen of 27 and 25 kDa). By indirect immunofluorescence with a monoclonal antibody 1H12 specific for Pfg 27/25, this protein is present in gametocytes within 30 to 40 hr after invasion of a red blood cell by a merozoite and is present throughout subsequent maturation of the gametocyte; Pfg 27/25 is not detectable on the surface of extracellular gametes by immunofluorescence with Mab 1H12. Pfg 27/25 is absent from asexual stages of P. falciparum at any stage in their development. Pfg 27/25 is an abundant protein in gametocytes and represents between 5 and 10% of total protein of these stages. Pfg 27/25 is also a major immunogen in man during P. falciparum infection. Antibodies to this protein were readily detected in human sera from an area of holoendemic P. falciparum and also from an individual following a primary attack of P. falciparum.

Nonspecific Immunoglobulin M Binding and Chondroitin Sulfate A Binding Are Linked Phenotypes of Plasmodium falciparum Isolates Implicated in Malaria during Pregnancy

ABSTRACT Binding of immunoglobulin M (IgM) antibodies from normal human serum to the surface of Plasmodium falciparum-infected red blood cells (iRBC) has previously been demonstrated only in parasites that form rosettes with uninfected red cells. We show that natural, nonspecific IgM but not IgG, IgA, IgD, or IgE also binds to the surface of iRBC selected for adhesion to chondroitin sulfate A (CSA), a placental receptor for parasites associated with malaria in pregnancy. The protease sensitivity of IgM-binding appears to match that of CSA binding, suggesting that the two phenotypes may be mediated by the same parasite molecule. We also show that a wide range of mouse monoclonal antibodies of the IgM class bind nonspecifically to CSA-selected iRBC, an important consideration in the interpretation of immunological assays performed on these parasite lines.

Maternal inheritance of extrachromosomal DNA in malaria parasites

Plasmodium falciparum has two extrachromosomal genomes, the mitochondrial 6-kb DNA element and the 35-kb circular DNA. The mitochondrial gene cytochrome b on the 6-kb element has been shown to be inherited uniparentally. In order to ascertain whether the route is maternal or paternal we have examined preparations of male and female gametes of the closely related Plasmodium gallinaceum for the presence of extrachromosomal DNA. DNA from purified preparations of gametes was hybridised to probes for both the 6-kb and 35-kb extrachromosomal genomes. Both probes hybridised to the preparation of Plasmodium gallinaceum female gametes but not to that of the males. We conclude that the extrachromosomal DNAs of malaria parasites are transmitted maternally.

Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

BackgroundClassical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.ResultsA lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina® Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.ConclusionsThis integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.

Uniparental inheritance of the mitochondrial gene cytochrome b in Plasmodium falciparum

The inheritance of an extrachromosomal 6-kb element has been examined in the human malaria parasite Plasmodium falciparum. A single base pair difference in the cytochrome b gene from the 6-kb element of two different cloned lines of the parasite was identified, and used as a marker in a cross in the mosquito stage of the life cycle. Analysis of 59 individual hybrid oocysts resulting from this cross clearly demonstrated that inheritance of the cytochrome b gene was uniparental. This observation makes it possible to investigate the inheritance and evolution of cytoplasmic traits, including certain forms of drug resistance, in natural populations of this parasite.

Eleven years of malaria surveillance in a Sudanese village highlights unexpected variation in individual disease susceptibility and outbreak severity.

An analysis is presented of continuous data collected over 11 years based on 1,902,600 person/days of observation on the malaria experience of the people of Daraweesh, a village in eastern Sudan. Malaria transmission is hypo-endemic: the acquisition of clinical immunity with age is not as obvious as in more holo-endemic areas and malaria remained a problem in all age groups throughout the study. However, this population, who are of Fulani origin, showed a distinctly variable level of disease susceptibility. Thirty-two percent of the village never reported malaria symptoms or required malaria treatment while others experienced up to 8 clinical episodes over the 11 years of observation. Malaria incidence was clearly influenced by drought but much less obviously by rainfall. To what extent outbreak patterns are explicable in terms of anopheline factors, and to human immune factors, remains an interesting question for malaria modelling in this, and in other low transmission zones, such as the burgeoning urban areas of modern Africa.

Comparative Testing of Six Antigen-Based Malaria Vaccine Candidates Directed Toward Merozoite-Stage Plasmodium falciparum

ABSTRACT Immunogenicity testing of Plasmodium falciparum antigens being considered as malaria vaccine candidates was undertaken in rabbits. The antigens compared were recombinant baculovirus MSP-119 and five Pichia pastoris candidates, including two versions of MSP-119, AMA-1 (domains I and II), AMA-1+MSP-119, and fused AMA-1/MSP-119). Animals were immunized with equimolar amounts of each antigen, formulated in Montanide ISA720. The specificities and titers of antibodies were compared using immunofluorescence assays and enzyme-linked immunosorbent assay (ELISA). The antiparasite activity of immunoglobulin G (IgG) in in vitro cultures was determined by growth inhibition assay, flow cytometry, lactate dehydrogenase assay, and microscopy. Baculovirus MSP-119 immunizations produced the highest parasite-specific antibody titers in immunofluorescence assays. In ELISAs, baculovirus-produced MSP-119 induced more antibodies than any other single MSP-119 immunogen and three times more MSP-119 specific antibodies than the AMA-1/MSP-119 fusion. Antibodies induced by baculovirus MSP-119 gave the highest levels of growth inhibition in HB3 and 3D7 parasite cultures, followed by AMA-1+MSP-119 and the AMA-1/MSP-119 fusion. With the FCR3 isolate (homologous to the AMA-1 construct), antibodies to the three AMA-1-containing candidates gave the highest levels of growth inhibition at high IgG concentrations, but antibodies to baculovirus MSP-119 inhibited as well or better at lower IgG concentrations. The two P. pastoris-produced MSP-119-induced IgGs conferred the lowest growth inhibition. Comparative analysis of immunogenicity of vaccine antigens can be used to prioritize candidates before moving to expensive GMP production and clinical testing. The assays used have given discriminating readouts but it is not known whether any of them accurately reflect clinical protection.