The genome of the simian and human malaria parasite Plasmodium knowlesi.

Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

Differential recognition patterns of Schistosoma haematobium adult worm antigens by the human antibodies IgA, IgE, IgG1 and IgG4.

Schistosoma haematobium antigen recognition profiles of the human isotypes IgA, IgE, IgG1 and IgG4 were compared by image analysis of western blots. Adult worm antigens separated by two-dimensional gel electrophoresis were probed with pooled sera from Zimbabweans resident in a S. haematobium endemic area, followed by the identification of individual antigenic parasite proteins using mass spectrometry. Overall, IgG1 reacted with the largest number of antigens, followed by IgE and IgA which detected the same number, while IgG4 detected the fewest antigens. IgE recognized all antigens reactive with IgG4 as well as an additional four antigens, an isoform of 28-kDa GST, phosphoglycerate kinase, actin 1 and calreticulin. IgG1 additionally recognized fatty acid-binding protein, triose-phosphate isomerase and heat shock protein 70, which were not recognized by IgA. Recognition patterns varied between some isoforms, e.g. the two fructose 1-6-bis-phosphate aldolase isoforms were differentially recognized by IgA and IgG1. Although the majority of S. haematobium adult worm antigens are recognized by all of the four isotypes, there are clear restrictions in antibody recognition for some antigens. This may partly explain differences observed in isotype dynamics at a population level. Differential recognition patterns for some isoforms indicated in the study have potential importance for vaccine development.

Analysis of a donor gene region for a variant surface glycoprotein and its expression site in African trypanosomes.

African trypanosomes evade the immune response of their mammalian hosts by sequentially expressing genes for different variant surface glycoproteins (VSGs) from telomere-linked VSG expression sites. In the Trypanosoma brucei clone whose genome is being sequenced (GUTat 10.1), we show that the expressed VSG (VSG 10.1) is duplicated from a silent donor VSG located at another telomere-linked site. We have determined two 130 kb sequences representing the VSG 10.1 donor and expression sites. The telomere-linked donor VSG 10.1 resembles metacyclic VSG expression sites, and is preceded by a cluster of 35 or more tandem housekeeping genes, all of which are transcribed away from the telomere. The 45 kb telomere-linked VSG 10.1 expression site contains a promoter followed by seven expression site-associated genes (ESAGs), three pseudo ESAGs, two pseudo VSGs and VSG 10.1. The 80 kb preceding the expression site has few, if any, functional ORFs, but contains 50 bp repeats, INGI retrotransposon-like elements, and novel 4-12 kb repeats found near other telomeres. This analysis provides the first look over a 130 kb range of a telomere-linked donor VSG and its corresponding telomere-linked VSG expression site and forms the basis for studies on antigenic variation in the context of a completely sequenced genome.

Linkage of the cholesteryl ester transfer protein (CETP) gene to LDL particle size: use of a novel tetranucleotide repeat within the CETP promoter.

BACKGROUND: A preponderance of small, dense LDL particles, elevated levels of plasma triglycerides (TG), and low levels of HDL characterize the atherogenic lipoprotein phenotype, which is associated with increased coronary artery disease (CAD) risk. Genetic and environmental factors influence LDL size, cholesteryl ester transfer protein (CETP) being one of the candidate genes. CETP mediates the transfer of cholesteryl ester from HDL to apolipoprotein (apo) B-containing lipoproteins in exchange for TG, promoting reverse cholesterol transfer and remodeling of lipoprotein particles. METHODS AND RESULTS: We have identified a tetranucleotide repeat (fragment sizes from 324 to 464 bp; heterozygosity index = 0.74) within the CETP promoter and used it in quantitative sib-pair linkage analysis in 119 female dizygotic (DZ) twins. Linkage was found to LDL size (P<0.001), TG (P<0.005), and plasma apoB (P = 0.02). The distribution of the tetranucleotide repeats was bimodal, and there was strong allelic association of the "short" alleles with the B2 allele of CETP TaqIB polymorphic site (P<0.001). CONCLUSIONS: This report of linkage of the CETP gene to LDL particle size adds to the list of candidate genes linked to LDL size, supporting the hypothesis of multigenic determination of LDL size heterogeneity. Whether this promoter variation is itself functional or is a marker for a functional site in the CETP gene remains to be determined.

Morphogenesis of Drosophila pupal wings in vitro.

We have developed an in vitro culture system which supports the differentiation of Drosophila pupal wings. Cultured wings develop marginal bristles and wing veins, and wing cells form a single prehair at their distal vertex at the appropriate developmental stages. We have tested two molecules with well defined activities to determine the usefulness of this system for applying pharmacological approaches to wing differentiation. Cycloheximide (CY) is a small molecule which inhibits protein synthesis. We found that 50 nM CY rapidly blocks all stages of wing differentiation without lowering cell viability. Chitinase is an enzyme which cleaves chitin polymers and is involved in normal cuticle apolysis. Chitinase applied prior to 28 h apf caused a contraction of the wing without affecting the general wing pattern. We have detected connections between the epithelium and pupal cuticle that are presumably targets of chitinase and are necessary for maintaining normal tissue shape during morphogenesis. Later in development exposure to chitinase caused a loss of normal prehair and bristle polarity, and high doses resulted in a severe disruption of the actin cytoskeleton.

Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila.

We have found that the actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine was able to slow prehair extension. At higher doses a complete blockage of hair development was seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. The multiple hair cells were a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development as treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton.

A high level of mixed Trypanosoma brucei infections in tsetse flies detected by three hypervariable minisatellites.

The issue of whether genetic exchange occurs at a significant frequency in natural populations of Trypanosoma brucei is controversial and one of the arguments against a high frequency has been the apparent lack of host infections with mixtures of trypanosome genotypes. Three minisatellite markers (MS42, CRAM, 292) within the coding regions of three genes have been identified and PCR based methods developed for detecting variation at these loci using crude lysates of infected blood as templates. Initial PCR analysis, using primers flanking the repeats, of DNA from two cloned stocks of the parasite has shown that two DNA fragments of different size were amplified from each stock. Analysis of the inheritance of these fragments into the F1 progeny of crosses demonstrated that the different size fragments were alleles that segregated in a Mendelian manner. The alleles at each of the three loci segregated independently consistent with their localisation on three different chromosomes. Analysis of a series of cloned isolates from tsetse flies showed that these loci were highly variable giving heterozygosities of 94% and the identification of 12 distinct alleles in a sample of 17 cloned isolates. In order to determine whether isolates are heterogeneous in terms of trypanosome genotype, the allelic variation at these three loci was examined in uncloned samples from tsetse flies isolated in Kiboko, Kenya and Lugala, Uganda. A significant proportion of the isolates (36% in Lugala and 47% in Kiboko) contained more than two alleles at one or more of the loci thus demonstrating that a high proportion of tsetse flies were infected with more than one genotype of trypanosomes. This was established, unequivocally, for two isolates by generating a series of cloned trypanosome lines from each and determining the genotype of each clone; one isolate (927) contained seven different genotypes with a high proportion of the possible combinations of alleles at each locus. These results indicate the possibility of frequent genetic exchange in the field, they imply that a significant proportion of mammalian hosts must contain mixtures of different trypanosome genotypes and they demonstrate the advantages of using minisatellite markers for the analysis of the population structure of T. brucei.

Self-fertilisation in Trypanosoma brucei.

We have investigated whether Trypanosoma brucei can undergo self-fertilisation. A group of 27 metacyclic clones derived from the tsetse transmission of a mixture of two genetically marked stocks was analysed and 22 clones were observed to be of non-hybrid phenotype. A group of 10 clones from this non-hybrid subset were then analysed for one isoenzyme, one restriction fragment length polymorphism and three karyotype markers potentially informative for the detection of self-fertilisation. Five of the 10 clones were found to be recombinant for at least one marker and we interpret these recombination events as indicating the clones to be products of self-fertilisation. We have also analysed a limited number of metacyclic clones from stocks of T. brucei each singly transmitted through tsetse flies but, so far, no evidence of recombination has been detected. We conclude that T. brucei is able to self-fertilise but there may be a requirement for the presence of dissimilar stocks to initiate such an event.

Genetic evidence that metacyclic forms of Trypanosoma brucei are diploid.

The hypothesis that metacyclic trypanosomes are haploid has been tested genetically. Five cloned stocks of Trypanosoma brucei (each having four known isoenzyme markers and six known restriction fragment length polymorphisms) have been independently transmitted through tsetse flies. Fifteen individual metacyclic organisms were taken from flies with mature cyclical infections and used to establish fresh clones. All the sub-clones from all the flies proved to be identical to the starting (parental) stocks, with respect to all the markers examined, including those markers which were heterozygous in the parental stocks. We conclude that metacyclic trypanosomes are diploid, and are not the product of an obligatory meiosis.

Analysis of ploidy (in megabase chromosomes) in Trypanosoma brucei after genetic exchange.

The megabase chromosomes of Trypanosoma brucei are normally diploid, but the extent to which this ploidy is maintained when parasites undergo genetic exchange is not known. To investigate this issue, a panel of 30 recombinant clones resulting from the co-transmission through tsetse flies of three different parental T. brucei lines in all pair-wise combinations (STIB 247, STIB 386 and TREU 927/4) were examined. These clones are products of 28 different mating events; four of them result from self-fertilisation and the others are F1 hybrids. DNA contents of the three parental lines were determined by flow cytometry and shown to differ only slightly with DNA content increasing in the order 927/4 < 247 < 386. Flow cytometry of the recombinant clones indicated DNA contents were similar to the parents in 28 clones and raised approximately 1.5 times the parental values in only two. The two F1 hybrid progeny with raised DNA contents were shown by marker analysis to be trisomic for seven independent loci indicating that they were probably triploid whereas progeny with DNA contents similar to parental values inherited a single allele from each parent for four independent loci indicating that they were diploid.

Gene discovery in Plasmodium chabaudi by genome survey sequencing.

The first genome survey sequencing of the rodent malaria parasite Plasmodium chabaudi is presented here. In 766 sequences, 131 putative gene sequences have been identified by sequence similarity database searches. Further, 7 potential gene families, four of which have not previously been described, were discovered. These genes may be important in understanding the biology of malaria, as well as offering potential new drug targets. We have also identified a number of candidate minisatellite sequences that could be helpful in genetic studies. Genome survey sequencing in P. chabaudi is a productive strategy in further developing this in vivo model of malaria, in the context of the malaria genome projects.

Characterisation of the growth and differentiation in vivo and in vitro-of bloodstream-form Trypanosoma brucei strain TREU 927.

Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete sequencing of the genome of the African trypanosome. This line is pleomorphic in mammalian hosts and is fly transmissible; however it is relatively unstable with respect to variable surface glycoprotein (VSG) expression. Therefore, we subjected TREU 927/4 to 27 rapid syringe passages through mice, and derived a cloned line which expressed Glasgow University Trypanozoon antigen type (GUTat) 10.1 with relative stability. This line also retained pleomorphism in the bloodstream, being able to generate homogeneous populations of stumpy forms in mice. Furthermore, these parasites remain able to transform to procyclic forms synchronously in vitro and can complete their life cycle in tsetse flies. The passaged cell line was also adapted to in vitro bloodstream-form culture and transfected with a construct encoding the tetracycline repressor (TETR) protein. The resulting TETR subline no longer expressed the GUTat 10.1 VSG but remained able to generate uniform populations of stumpy form cells in mice immunocompromised with cyclophosphamide. They could also differentiate to procyclic forms synchronously in vitro. The generated lines and analyses of their growth and differentiation will provide a basic resource for the analysis and interpretation of gene function in the T. brucei genome reference strain.

Gene exchange in African trypanosomes: frequency and allelic segregation.

The existence of a system of genetic exchange in Trypanosoma brucei is now established, but the frequency with which mating occurs and the mechanisms by which genes are exchanged are still unknown. This paper presents the results of a study of one pair of trypanosome stocks, which show that mating is a non-obligatory but frequent event in a life-cycle stage within the insect vector. Analysis of ten progeny clones using a total of eleven markers (iso-enzymes and DNA probes detecting restriction fragment length polymorphisms) has indicated that segregation of alleles occurs at five of these loci. The segregation patterns of a polymorphic EcoRI site in the maxi-circle of the kinetoplast DNA (kDNA) show that the progeny inherit one or other of the parental kDNA types. These results demonstrate that segregation of alleles occurs and that new combinations of alleles at different loci are generated in the progeny clones. The implications of these findings for defining the mechanism of gene exchange are discussed in relation to a simple mendelian genetic system involving meiosis and syngamy.

Malaria parasites undergo antigenic variation at high rates in vivo.

The rates of switching between expression of variable antigen types (VATS) have been investigated in vivo in a cloned line of Plasmodium chabaudi chabaudi. VAT-specific hyperimmune sera combined with an immunogold-silver staining technique were used to detect VATS, and five estimates of VAT-specific switching rates were determined for three of them. VAT-specific switching rates were consistent for each VAT and differed between VATS in the range 1.3 x 10(-2) to 4.3 x 10(-4) switches per schizont per generation. This variation suggests that hierarchical expression of VATS in an infection may be determined by switch rates. A minimum estimate of the overall switching rate was determined by summation of the VAT-specific rates in each of two experiments. In both cases the results showed that at least 1 in every 80 schizonts switched VAT expression every generation. This is the first report of antigenic switching rates for malaria parasites measured in vivo in the presence of minimal specific immune pressure, and is the first to show that VAT-specific switching rates vary between VATS. We conclude that switching is rapid and spontaneous, and is regulated, at least in part, by the VATS involved.

Contrasting cellular responses in Schistosoma haematobium infected and exposed individuals from areas of high and low transmission in Zimbabwe.

The study compared cytokine profiles of individuals from two areas with different transmission patterns for Schistosoma haematobium. One area was a high transmission (HT) while the other was a low transmission (LT) area for S. haematobium. Observations on cellular immune responses were made on stimulated peripheral blood mononuclear cells (PBMC), which were collected pre-treatment, then at 12 and 18 months post treatment. Stimulation was with schistosome worm and egg antigens and a mitogen, phaetohaemaglutinin (PHA). Observations were made on PBMC proliferation and the profiles of cytokine produced over a 5-day incubation period. The two distinct areas showed significant differences on both levels of proliferation and cytokine production for all the measured classes (IL-4, IL-5, IL-10 and IFN-gamma). PBMC from individuals from the LT area had high levels of proliferation but low cytokine production to both antigen stimulants while PBMC from individuals from the HT area showed low levels of proliferation but high cytokine production levels. Prior to treatment, individuals not excreting schistosome ova in the HT area had higher levels of proliferation to the stimulants, than the infected individuals. However, after treatment re-infected individuals showed high levels of proliferation. Before treatment, both infected and uninfected groups showed low and similar ratios, respectively, of IL-4:IFN-gamma, IL-5:IFN-gamma and IL-10:IFN-gamma, while IFN-gamma was high in the infected individuals. After treatment the non re-infected had higher levels of IL-4, IL-5 and IL-10, with the infected having high levels of IFN-gamma. Th1-like response dominated during infection with the Th2-like responses dominating post treatment and in uninfected individuals. The results indicated that the cytokine balance determines, in part, susceptibility or resistance to S. haematobium infection.

The rate of antigenic variation in fly-transmitted and syringe-passaged infections of Trypanosoma brucei.

Rates of antigenic variation were measured in vivo in several populations of cloned lines of Trypanosoma brucei before and after cyclical transmission through tsetse flies. Two cloned lines were adapted for use in laboratory conditions by extensive syringe passaging and rates of antigenic switching/cell/generation were less than 3 x 10(-6) and 1 x 10(-4) in each line. Rates of switching were then determined after fly transmission of the first line and generated per capita rate values of greater than 2 x 10(-3) in three of four populations examined. In the fourth population the switch rate was lower: less than 7 x 10(-5) switches/ cell/generation. These data show that rates of antigenic variation are several orders of magnitude lower in syringe-passaged lines, such as those routinely used in the majority of laboratory studies, compared with most recently fly-transmitted lines. They also show that the reduction in switching rate associated with syringe passaging is reversible and is thus unlikely to be caused by mutation.

Similarity in variable antigen type composition of Trypanosoma brucei rhodesiense populations in different sites within the mouse host.

Trypanosoma brucei rhodesiense subpopulations in different sites within the body of infected mice were isolated and enumerated on day 6 of cyclically transmitted infections. Most trypanosomes were in the blood vasculature and spleen but approximately 6% occurred in lymph nodes and about 9% were extravascular. Most of the extravascular trypanosomes were in the peritoneal and pleural cavities; significant numbers also occurred in the brain and kidneys. Six major variable antigen types (VATs) were detected by immunofluorescence using specific antisera and monoclonal antibodies. The prevalence of each VAT was essentially the same in subpopulations in the blood, mesenteric and inguinal lymph nodes, brain, kidneys and peritoneal and pleural cavities. This similarity of VAT composition in different subpopulations is probably caused by high rates of dynamic interchange of trypanosomes between sites. Extravascular trypanosomes, therefore, form a significant proportion of the total population in acute infections of mice but they do not appear to play any special role in the population biology of antigenic variation at this stage of infection.

Characterisation of cloned lines of Trypanosoma brucei expressing stable resistance to MelCy and suramin.

Two cloned drug-sensitive stocks of Trypanosoma brucei (STIB 247 and STIB 386) were each used to generate cloned lines expressing resistance to the melaminophenyl arsenical drug cymelarsan (247MelCyR and 386MelCyR) and to suramin (247SurR and 386SurR). The drug-resistance phenotypes were stable after passaging in mice in the absence of drug pressure and three of the lines were transmitted through tsetse flies with no alteration of drug-resistance. There was no evidence of cross-resistance between melCy and suramin in vivo. Twenty-four hour growth inhibition assays were conducted on bloodstream and procyclic forms in axenic in vitro cultures. Suramin-resistance was expressed in bloodstream forms but not in the procyclic stage, while the melCy-resistant lines expressed melCy-resistance in both stages. No cross-resistance between melCy and suramin was observed. Cross-resistance between melCy and another arsenical drug, melB (melarsoprol), was observed in vivo, but to only a very limited extent in vitro. We propose that this difference between the in vivo and in vitro results for melB may indicate that an alteration in a surface adenosine transporter responsible for reduced melCy uptake was bypassed by melB over 24 hours in vitro.

Metabolism and distribution of phenanthridine trypanocides in Trypanosoma brucei.

Phenanthridine trypanocides (isometamidium chloride hydrochloride, ISM, and Ethidium bromide, EBr) have been widely used to treat African trypanosomiasis in livestock for more than 40 years. Their main action is to inhibit nucleic acid synthesis in trypanosome parasites, by intercalation between the DNA base pairs. They can also linearise selectively kinetoplast DNA minicircles; a form of mitochondrial DNA unique to this group of parasites. However, the metabolism of these compounds by trypanosomes has not been reported. Indeed, it is not known whether or not their metabolism by the parasite contributes to their activity, selective toxicity for these parasites or to the development of chemoresistance. Therefore, we studied the metabolism of EBr and ISM, and their distribution in Trypanosoma brucei (TREU 927) using high performance liquid chromatography (HPLC), liquid chromatography combined with mass spectrometry (LC-MS) and confocal laser scanning microscopy (CLSM). Incubation of EBr with trypanosomes led to the formation of a small amount (0.606+/-0.191%) of one metabolite (MI). Ion chromatograms extracted from an LC-MS analysis using electrospray ionisation (ESI), showed that the difference in mass between the parent compound and its metabolite was 30. This may correspond to the addition of a hydroxyl and a methyl group. No metabolites could be detected for ISM. The distribution of the two drugs in trypanosomes was investigated by CLSM, using their intrinsic fluorescence. ISM and EBr showed differences in their distribution in trypanosomes. ISM had a greater affinity for the kinetoplast than EBr and it stained other organelles like the flagellum; in contrast the distribution of EBr was more diffuse.

P2Y receptors present in the native and isolated rat glomerulus.

Extracellular ATP can mobilize intracellular calcium in rat glomeruli by interacting with P2Y receptors. However, the identity of the receptor subtypes involved is not known. In the present study, we have used RT-PCR to identify mRNAs for specific P2Y receptor subtypes expressed in the rat glomerulus: mRNA for P2Y1, P2Y2, P2Y4 and P2Y6 receptors was detected. Functional expression of P2Y1 and P2Y2/P2Y4, but not P2Y6, receptors in intact glomeruli was confirmed by measuring the relative stimulation of the inositol phosphate pathway induced by selective agonists of a particular receptor subtype. Finally, we have used available polyclonal antibodies to confirm the expression of P2Y1 and P2Y2 in the glomerulus, in mesangial cells and glomerular epithelial cells (podocytes), respectively; but we could not demonstrate P2Y4 or P2Y6 receptor expression by this means. In a separate series of experiments, we have examined the possibility that intra-renal sympathetic nerve terminals are a source of extracellular ATP and that this would be supported, though not excluded, by supersensitivity to ATP following denervation. Nucleotide-induced stimulation of the inositol phosphate pathway was measured in both control rats and rats that had been sympathectomized by intraperitoneal injection of 6-hydroxydopamine. The response to norepinephrine was measured as a positive control. In the sympathectomized rats, the effect of norepinephrine was significantly enhanced, whereas ATP-induced inositol phosphate production was unaffected, being similar in both groups of animals.