Selection of Plasmodium falciparum multidrug resistance gene 1 alleles in asexual stages and gametocytes by artemether-lumefantrine in Nigerian children with uncomplicated falciparum malaria.

We assessed Plasmodium falciparum mdr1 (Pfmdr1) gene polymorphisms and copy numbers as well as P. falciparum Ca(2+) ATPase (PfATPase6) gene polymorphisms in 90 Nigerian children presenting with uncomplicated falciparum malaria and enrolled in a study of the efficacy of artemether-lumefantrine (AL). The nested PCR-restriction fragment length polymorphism and the quantitative real-time PCR methodologies were used to determine the alleles of the Pfmdr1 and PfATPase6 genes and the Pfmdr1 copy number variation, respectively, in patients samples collected prior to treatment and at the reoccurrence of parasites during a 42-day follow-up. The Pfmdr1 haplotype 86N-184F-1246D was significantly associated (P < 0.00001) with treatment failures and was selected for among posttreatment samples obtained from patients with newly acquired or recrudescing infections (P < 0.00001; chi(2) = 36.5) and in gametocytes (log rank statistic = 5; P = 0.0253) after treatment with AL. All pre- and posttreatment samples as well as gametocytes harbored a single copy of the Pfmdr1 gene and the wild-type allele (L89) at codon 89 of the PfATPase6 gene. These findings suggest that polymorphisms in the Pfmdr1 gene are under AL selection pressure. Pfmdr1 polymorphisms may result in reduction in the therapeutic efficacy of this newly adopted combination treatment for uncomplicated falciparum malaria in Saharan countries of Africa.

Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane.

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.

Leishmaniasis and malaria: new tools for epidemiologic analysis.

Parasitic diseases are still prevalent in many parts of the world, causing both human suffering and economic loss. Recent developments in biotechnology, such as the use of monoclonal antibodies and recombinant DNA, have the potential for providing both more extensive and detailed information on the parasite in the infected human and in insect vectors. New methods of detection, both in man and insect vectors, have been developed for two parasitic diseases, leishmaniasis and malaria. These new methodologies will be important in epidemiologic studies on the prevalence and transmission of these parasitic diseases.

Specific DNA probe for the diagnosis of Plasmodium falciparum malaria.

Malaria can be diagnosed either by direct microscopic examination of blood smears, which is time consuming and requires expertise, or by immunological techniques, which are effective but do not distinguish between past and present infections. In this study, a simple procedure was developed for spotting lysed blood from infected patients directly onto nitrocellulose paper and identifying the malaria species on the basis of hybridization of parasite DNA with a species-specific probe. A genomic DNA library of Plasmodium falciparum was screened to detect clones containing DNA sequences that are highly repeated within the parasite genome. Several such clones were further analyzed to identify those that hybridize specifically with P. falciparum DNA but not with DNA from humans, P. vivax, or P. cynomolgi. This technique appears to be sensitive enough to detect 10 picograms of purified P. falciparum DNA (equivalent to 100 parasites) and in field studies is able to detect approximately 40 parasites per microliter of blood.

Amplification of a gene related to mammalian mdr genes in drug-resistant Plasmodium falciparum.

The malaria parasite Plasmodium falciparum contains at least two genes related to the mammalian multiple drug resistance genes, and at least one of the P. falciparum genes is expressed at a higher level and is present in higher copy number in a strain that is resistant to multiple drugs than in a strain that is sensitive to the drugs.

Recent origin of Plasmodium falciparum from a single progenitor.

Genetic variability of Plasmodium falciparum underlies its transmission success and thwarts efforts to control disease caused by this parasite. Genetic variation in antigenic, drug resistance, and pathogenesis determinants is abundant, consistent with an ancient origin of P. falciparum, whereas DNA variation at silent (synonymous) sites in coding sequences appears virtually absent, consistent with a recent origin of the parasite. To resolve this paradox, we analyzed introns and demonstrated that these are deficient in single-nucleotide polymorphisms, as are synonymous sites in coding regions. These data establish the recent origin of P. falciparum and further provide an explanation for the abundant diversity observed in antigen and other selected genes.

Erythrocyte invasion profiles are associated with a common invasion ligand polymorphism in Senegalese isolates of Plasmodium falciparum.

Plasmodium falciparum parasites use multiple ligand-receptor interactions to invade human erythrocytes. Variant expression levels of members of the PfRh and PfEBA ligand families are associated with the use of different erythrocyte receptors, defining invasion pathways. Here we analyse a major polymorphism, a large sequence deletion in the PfRh2b ligand, and erythrocyte invasion profiles in uncultured Senegalese isolates. Parasites vary considerably in their use of sialic acid-containing and protease-sensitive erythrocyte receptors for invasion. The erythrocyte selectivity index was not related to invasion pathway usage, while parasite multiplication rate was associated with enhanced use of a trypsin-resistant invasion pathway. PfRh2b protein was expressed in all parasite isolates, although the PfRh2b deletion was present in a subset (approximately 68%). Parasites with the PfRh2b deletion were found to preferentially utilize protease-resistant pathways for erythrocyte invasion. Sialic acid-independent invasion is reduced in parasites with the PfRh2b deletion, but only in isolates derived from blood group O patients. Our results suggest a significant role for PfRh2b sequence polymorphism in discriminating between alternative erythrocyte receptors for invasion and as a possible determinant of virulence.

trans splicing in Leishmania enriettii and identification of ribonucleoprotein complexes containing the spliced leader and U2 equivalent RNAs.

The 5' ends of Leishmania mRNAs contain an identical 35-nucleotide sequence termed the spliced leader (SL) or 5' mini-exon. The SL sequence is at the 5' end of an 85-nucleotide primary transcript that contains a consensus eucaryotic 5' intron-exon splice junction immediately 3' to the SL. The SL is added to protein-coding genes immediately 3' to a consensus eucaryotic 3' intron-exon splice junction. Our previous work demonstrated possible intermediates in discontinuous mRNA processing that contain the 50 nucleotides of the SL primary transcript 3' to the SL, the SL intron sequence (SLIS). These RNAs have a 5' terminus at the splice junction of the SL and the SLIS. We examined a Leishmania nuclear extract for these RNAs in ribonucleoprotein (RNP) particles. Density centrifugation analysis showed that the SL RNA is predominantly in RNP complexes at 60S, while the SLIS-containing RNAs are in complexes at 40S. We also demonstrated that the SLIS can be released from polyadenylated RNA by incubation with a HeLa cell extract containing debranching enzymatic activity. These data suggested that Leishmania enriettii mRNAs are assembled by bimolecular or trans splicing as has been recently demonstrated for Trypanosoma brucei. Furthermore, we determined the partial sequence of the Leishmania U2 equivalent RNA and demonstrated that it cosediments with the SL RNA at 60S in a nuclear extract. These RNP particles may be analogous to so-called spliceosomes that have been demonstrated in other systems.

Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

Sequence identification of cytochrome b in Plasmodium gallinaceum.

We have identified a gene that encodes the polypeptide cytochrome b in the avian malarial parasite Plasmodium gallinaceum. The gene containing the open reading frame was found to be located on a 6.2-kilobase multimeric extrachromosomal element. The amino acid translation from this gene demonstrated significant similarities to cytochrome b sequences from yeast, mammal, and fungus genomes. We present evidence that the P. gallinaceum cytochrome b transcript is part of a larger primary transcript from the element that is subsequently processed. The message for P. gallinaceum cytochrome b was found to be 1.2 kilobases in size. This is the first report identifying a mitochondrial nucleic acid sequence in malaria-causing organisms and suggests that a functional cytochrome system may exist in these parasites.

Characterization of a conserved extrachromosomal element isolated from the avian malarial parasite Plasmodium gallinaceum.

We have identified a conserved, repeated, and highly transcribed DNA element from the avian malarial parasite Plasmodium gallinaceum. The element produced multiple transcripts in both zygotes and asexual blood stages of this parasite. It was found to be highly conserved in all of five malarial species tested and hybridized at reduced stringency to other members of the phylum Apicomplexa, including the genera Babesia, Eimeria, Toxoplasma, and Theileria. The copy number of the element was about 15, and it had a circularly permuted restriction map with a repeat unit length of about 6.2 kilobases. It could be separated from the main genomic DNA by using sucrose gradients and agarose gels, and it migrated separately from the recognized Plasmodium chromosomes on pulse-field gels. In the accompanying paper (S. M. Aldritt, J. T. Joseph, and D. F. Wirth, Mol. Cell. Biol. 9:3614-3620, 1989), evidence is presented that element contains the mitochondrial genes for the protein cytochrome b and a fragment of the large rRNA. We postulate that this element is an episome in the mitochondria of the obligate parasites belonging to the phylum Apicomplexa.

Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene.

Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.

Isolation and functional characterization of two distinct sexual-stage-specific promoters of the human malaria parasite Plasmodium falciparum.

Transmission of malaria depends on the successful development of the sexual stages of the parasite within the midgut of the mosquito vector. The differentiation process leading to the production of the sexual stages is delineated by several developmental switches. Arresting the progression through this sexual differentiation pathway would effectively block the spread of the disease. The successful development of such transmission-blocking agents is hampered by the lack of a detailed understanding of the program of gene expression that governs sexual differentiation of the parasite. Here we describe the isolation and functional characterization of the Plasmodium falciparum pfs16 and pfs25 promoters, whose activation marks the developmental switches executed during the sexual differentiation process. We have studied the differential activation of the pfs16 and pfs25 promoters during intraerythrocytic development by transfection of P. falciparum and during gametogenesis and early sporogonic development by transfection of the related malarial parasite P. gallinaceum. Our data indicate that the promoter of the pfs16 gene is activated at the onset of gametocytogenesis, while the activity of the pfs25 promoter is induced following the transition to the mosquito vector. Both promoters have unusual DNA compositions and are extremely A/T rich. We have identified the regions in the pfs16 and pfs25 promoters that are essential for high transcriptional activity. Furthermore, we have identified a DNA-binding protein, termed PAF-1, which activates pfs25 transcription in the mosquito midgut. The data presented here shed the first light on the details of processes of gene regulation in the important human pathogen P. falciparum.

Serial analysis of gene expression in Plasmodium falciparum reveals the global expression profile of erythrocytic stages and the presence of anti-sense transcripts in the malarial parasite.

Serial analysis of gene expression (SAGE) was applied to the malarial parasite Plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages. A SAGE library of approximately 8335 tags representing 4866 different genes was generated from 3D7 strain parasites. Basic local alignment search tool analysis of high abundance SAGE tags revealed that a majority (88%) corresponded to 3D7 sequence, and despite the low complexity of the genome, 70% of these highly abundant tags matched unique loci. Characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions. Furthermore several tags expressed at high abundance (30% of tags matching to unique loci of the 3D7 genome) were derived from previously uncharacterized open reading frames, demonstrating the use of SAGE in genome annotation. The open platform "profiling" nature of SAGE also lead to the important discovery of a novel transcriptional phenomenon in the malarial pathogen: a significant number of highly abundant tags that were derived from annotated genes (17%) corresponded to antisense transcripts. These SAGE data were validated by two independent means, strand specific reverse transcription-polymerase chain reaction and Northern analysis, where antisense messages were detected in both asexual and sexual stages. This finding has implications for transcriptional regulation of Plasmodium gene expression.

Association between mutations in Plasmodium falciparum chloroquine resistance transporter and P. falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in P. falciparum malaria-infected children in Nigeria.

This study investigated the association between Plasmodium falciparum chloroquine resistance transporter (pfcrt) T76 and P. falciparum multidrug resistance gene 1 (pfmdr1) Y86 alleles and in vivo amodiaquine (AQ) resistance, as well as the clearance of parasites harboring these two alleles in children treated with AQ in southwest Nigeria. One hundred one children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of AQ and followed-up for 28 days. Blood samples were collected on filter paper samples at enrollment and during follow-up for identification of parasite genotypes and pfcrt and pfmdr1 mutations using polymerase chain reaction and restriction fragment length polymorphism approaches. Parasitologic assessment of response to treatment showed that 87% and 13% (RI) of patients were cured and failed treatment, respectively. Although infections in patients were polyclonal (as determined by merozoite surface protein 2 genotyping), the presence of both mutants pfcrtT76 and pfmdr1Y86 alleles in parasites is associated with in vivo AQ resistance (odds ratio = 7.58, 95% confidence interval = 1.58-36.25, P = 0.006) and is selected by the drug in children who failed AQ treatment. Treatment failure with the combination of mutant pfcrtT76 and pfmdr1Y86 alleles as well as the ability of patients to clear these resistant parasites is dependent on age, suggesting a critical role of host immunity in clearing AQ-resistant P. falciparum. The combination of mutant pfcrtT76 and pfmdr1Y86 alleles may be useful markers for monitoring the development and spread of AQ resistance, when combining this drug with other antimalarials for treatment of malaria in Africa.

Defining the origin of Plasmodium falciparum resistant dhfr isolates in Senegal.

We previously reported a high baseline prevalence of mutations in the dhfr and dhps genes of Plasmodium falciparum throughout Senegal. The highest prevalence of the triple dhfr pyrimethamine associated mutations were found in isolates obtained in the western part of the country near the capital city of Dakar. In this study, we sought out to determine the relatedness of dhfr wild type and mutated strains by analyzing three microsatellite regions upstream of the dhfr locus. Twenty-six of the 31 wild type strains had a unique microsatellite pattern. In contrast, of the 17 isolates containing the triple mutation in dhfr, 11 had an identical microsatellite pattern. Diverse geographical isolates in Senegal containing the triple dhfr mutation have arisen from a limited number of ancestral strains. In addition, we demonstrate that these isolates have shared ancestry with the previously reported triple mutation haplotype found in Tanzania, South Africa, and southeast Asia. This common ancestry may have implications for the malaria control strategy for reducing the spread of sulfadoxine-pyrimethamine resistance in Senegal and elsewhere in Africa.

Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine-pyrimethamine resistance in malaria-infected patients from Nigeria.

Mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes have been used as means to predict treatment failure to sulfadoxine-pyrimethamine (SP) and for monitoring/surveillance of resistance to the drug in many areas where malaria is endemic. However, patients responses to treatment are significantly dependent on factors like host immunity profile of treated patients. In order to investigate the relationship between molecular markers of SP resistance, host immunity and clinical outcome, the association between pre-treatment dhfr and dhps genotypes, age and treatment outcomes was evaluated in 109 children treated with SP for acute uncomplicated malaria in Ibadan, Nigeria. Seventy-three percent of the children were cured with the drug, while 27% failed treatment after 28 days of follow-up. All children infected with parasites harboring less than two dhfr/dhps mutations were cured with SP. The dhfr triple (Asn-108/Ile-51/Arg-59) mutants or the dhps double mutants (Gly-437/Glu-540) were independently associated with SP treatment failure in children aged less than 5 years, but not in older children. The dhfr and dhps quintuple mutant (dhfr triple mutant+dhps double mutant) was the genotype most strongly associated with SP treatment failure (OR=24.72, 95%CI=8.24-74.15) in both younger and older children.

Mutational analysis of a signal sequence required for protein secretion in Leishmania major.

In eukaryotes, amino-terminal extensions, signal sequences, mediate the translocation of lysosomal, membrane and secreted proteins into the lumen of the endoplasmic reticulum (ER). Structure/function studies indicate that eukaryotic signal sequences are composed of 3 distinct domains, a positively charged amino-terminal domain, a central hydrophobic domain, and a polar carboxy-terminal domain. In an attempt to better understand protein trafficking in Leishmania we have constructed strains of Leishmania major that secrete an exogenous protein, interferon-gamma (IFN-gamma), under the control of a mammalian signal sequence. In this report we present a mutational analysis of this signal sequence. Deletion of the entire signal sequence or the hydrophobic core region prevents secretion of IFN-gamma and results in cytoplasmic expression of the protein. Mutations in the amino-terminal domain indicate that a net positive charge is not required for efficient secretion of IFN-gamma. Mutations in the carboxy-terminal domain are more complex and display two phenotypes, either they prevent expression of IFN-gamma or they have no effect on protein secretion. These results indicate that the function of the signal sequence in targeting proteins to the ER in Leishmania is similar to that observed in yeasts and higher eukaryotes and suggests that the Leishmania protein secretory apparatus may also be similar.

Generation of sequence diversity in the kinetoplast DNA minicircles of Leishmania mexicana amazonensis.

In order to understand the mechanisms which generate minicircle sequence diversity, we sequenced three minicircles belonging to the same or closely related sequence classes from the kinetoplast DNA of Leishmania mexicana amazonensis strains, PH8, Raimundo, and Josefa. Closely related minicircles from PH8 and Raimundo were unexpectedly found to differ at 11% of positions within the evolutionarily conserved region, but at only 3.9% of positions in the variable region. It thus appears that accumulation of point mutations will not account for the wide intra-strain and intra-subspecies divergence of the variable region. Comparison of more distantly related minicircles from PH8 and Josefa revealed only two short stretches of 70% homology within the variable region. These stretches of homology are not located in the same positions relative to the conserved regions in their respective minicircles. They may represent vestiges of recombinational events responsible for the rapid divergence of minicircle variable regions.

Identification of visceral Leishmania species with cloned sequences of kinetoplast DNA.

Several efforts have been made in order to develop more precise and sensitive methods in the identification of Leishmania parasites. We report here the identification of cloned subfragments of minicircle kinetoplast DNA (kDNA) isolated from L. donovani, WR352, which show different taxonomic specificities. Analysis of these fragments demonstrates a significant sequence diversity within the kDNA minicircle. For example, one cloned fragment was found to be present in all visceral Leishmania species tested, but was not present in any of the cutaneous Leishmania species. Another cloned fragment was only found in the strain from which it had been derived, and was not present in any of the other strains tested. In similar experiments with the New World visceral leishmanias (L. chagasi, WR518) several different cloned kDNA fragments were found to react with all of isolates of the L. chagasi tested, but not with any cutaneous Leishmania species, either from the Old World or the New World. It is of interest to note that these cloned L. chagasi kDNA fragments reacted with isolates of African visceral Leishmania species but not with isolates from India.