A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle

Green fluorescent protein (GFP) is a well-established reporter protein for the examination of biological processes. This report describes a recombinant Plasmodium berghei, PbGFPCON, that constitutively expresses GFP in a growth responsive manner in its cytoplasm from a transgene that is integrated into the genome and controlled by the strong promoter from a P. berghei elongation factor-1alpha gene. All life cycle forms of PbGFPCON except for male gametes can be easily visualized by fluorescent microscopy. PbGFPCON showed similar growth characteristics to wild type P. berghei parasites throughout the whole life cycle and can therefore be used as a reference line for future investigations of parasite-host cell interactions. The principle of automated fluorescence-based counting and sorting of live parasites from host cell backgrounds and different parasite forms from complex mixtures such as asynchronous blood stages is established. PbGFPCON allows the visualization and investigation of live parasite stages that are difficult and labor-intensive to observe, such as the liver and mosquito stages. PbGFPCON can be employed to establish the phenotype of independent mutant parasites. With the recent development of a second, independent selectable marker in P. berghei, PbGFPCON is a useful tool to investigate the effect of further genetic modifications on host-parasite interactions.     

Knockout of the rodent malaria parasite chitinase pbCHT1 reduces infectivity to mosquitoes

During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium. Plasmodium chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite, Plasmodium berghei. We show that the gene, named PbCHT1, is a structural ortholog of PgCHT1 of the avian malaria parasite Plasmodium gallinaceum and a paralog of PfCHT1 of the human malaria parasite Plasmodium falciparum. Targeted disruption of PbCHT1 reduced parasite infectivity in Anopheles stephensi mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds-an artificial situation where midgut invasion occurs before PM formation-suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that P. berghei ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution of Plasmodium-mosquito interactions.     

Stage-specific expression of aldolase isoenzymes in the rodent malaria parasite Plasmodium berghei.

We have cloned two gene (aldo-1 and aldo-2) encoding the glycolytic enzyme aldolase of the rodent malaria parasite Plasmodium berghei. The amino acid sequence of one gene product, ALDO-1, is virtually identical to P. falciparum aldolase whereas ALDO-2, the second gene product, is different and has 13% sequence diversity to ALDO-1. We expressed ALDO-2 as an active enzyme in Escherichia coli and compared the biochemical and kinetic properties to that of P. falciparum recombinant aldolase (ALDO-1 type). Based on the Km and Vmax constants for FMP and FBP, neither ALDO-1 nor ALDO-2 can be clearly assigned to any of the known mammalian isoenzyme classes. We demonstrate that expression of the two isoenzymes is developmentally regulated: specific antibody probes detect ALDO-1 in sporozoite stages of P. berghei and ALDO-2 is found in blood stage parasites.     

Green fluorescent protein as a marker in Plasmodium berghei transformation

We present a new marker that confers both resistance to pyrimethamine and green fluorescent protein-based fluorescence on the malarial parasite Plasmodium berghei. A single copy of the cassette integrated into the genome is sufficient to direct fluorescence in parasites throughout the life cycle, in both its mosquito and vertebrate hosts. Erythrocyte stages of the parasite that express the marker can be sorted from control parasites by flow cytometry. Pyrimethamine pressure is not necessary for maintaining the cassette in transformed parasites during their sporogonic cycle in mosquitoes, including when it is borne by a plasmid. This tool should thus prove useful in molecular studies of P. berghei, both for generating parasite variants and monitoring their behavior.     

Differential gene expression in the ookinete stage of the malaria parasite Plasmodium berghei

Plasmodium, the malaria parasite, undergoes a complex developmental program in its mosquito vector. The ookinete is the parasite form which invades the mosquito midgut and is an important stage for genetic mixing. To identify genes expressed during ookinete development and mosquito midgut invasion, purified zygotes and ookinetes of the rodent parasite Plasmodium berghei were used to construct a suppression subtractive hybridization cDNA library, enriched in sequences expressed in the ookinete stage. In addition to four genes coding for previously described major ookinete-secreted proteins, we isolated ookinete-expressed sequences representing 18 predicted genes. Their gene products include proteins involved in signal transduction and regulatory processes. For six of these genes our analysis provides the first evidence for expression in the ookinete stage. A majority of the genes are not expressed in the zygote, the preceding developmental stage. Furthermore, four of the genes are also transcribed in sporozoites, and one of these in merozoites, suggesting that they code for proteins with a function common to Plasmodium invasive stages.     

PTRAMP; a conserved Plasmodium thrombospondin-related apical merozoite protein

A gene encoding a 352 amino acid protein with a putative signal sequence, transmembrane domain and thrombospondin structural homology repeat was identified in the genome of the human malaria parasite, Plasmodium falciparum and the rodent malaria parasite, Plasmodium berghei. The protein localises in the apical organelles of P. falciparum and P. berghei merozoites within intraerythrocytic schizonts and has, therefore, been termed the Plasmodium thrombospondin-related apical merozoite protein (PTRAMP). PTRAMP co-localises with the Apical Merozoite Antigen-1 (AMA-1) in developing micronemes and subsequently relocates onto the merozoite surface. Although the gene appears to be specific to the Plasmodium genus, orthologues are present in the genomes of all malaria parasite species examined suggesting a conserved function in host-cell invasion. PTRAMP, therefore, has all the features to merit further evaluation as a malaria vaccine candidate.     

Primary sequences of two small subunit ribosomal RNA genes from Plasmodium falciparum

We have determined the complete sequence of two structurally distinct 18S ribosomal RNA genes from the malarial parasite Plasmodium falciparum. S1 nuclease analyses demonstrate that only one of the genes is represented in stable rRNA populations isolated from blood-stage parasites. Comparisons of homologous rRNA genes from Plasmodium berghei and P. falciparum reveal that they are identical at 86% of their positions. From comparisons of the Plasmodium genes to that of humans, it was possible to design genus-specific as well as species-specific oligonucleotide probes that can be used to distinguish the parasite 18S ribosomal RNA from that of its host. The utilization of these probes as diagnostic reagents is discussed.     

Oxidant defense enzymes of Plasmodium falciparum

We have measured and characterized three oxidant defense enzymes in early and late intraerythrocytic stages of the human malarial parasite, Plasmodium falciparum. Isolated early intraerythrocytic stages contain catalase (24.1 mumol min-1 (mg protein)-1) and superoxide dismutase (SOD; 6.3 units (mg protein)-1) but little or no glutathione peroxidase (GPX; less than 2 mumol min-1 (mg protein)-1). Isolated late intraerythrocytic stages of P. falciparum contain slightly less catalase (17.0 mumol min-1 (mg protein)-1) but significantly more GPX (7.7 mumol min-1 (mg protein)-1) and SOD (25.1 units (mg protein)-1). P. falciparum, like P. berghei, probably acquires most of its SOD from its host, since parasite-associated SOD is predominantly cyanide-sensitive, and has the same pI as host SOD. Unlike P. berghei, however, late stages of P. falciparum contain an additional SOD isozyme which is not cyanide-sensitive and may represent an endogenous enzyme. Parasites grown in red cells that have been partially depleted of SOD are more sensitive to exogenously generated superoxide, suggesting some dependence of the parasite on host SOD.     

Improved Plasmodium berghei lines for conditional mutagenesis

Conditional mutagenesis is a powerful tool for genetic analysis in Plasmodium berghei. It allows the study of proteins that function both during the parasite's pre-erythocytic and erythrocytic development. Currently available parasite lines used for conditional mutagenesis were constructed in the NK65 strain, and express a DNA recombinase under the control of pre-erythrocytic stage-specific promoters. However, the integration of the recombinase in these lines is unstable leading to inconsistent excision of the target gene. We describe improved lines of P. berghei with stably integrated DNA recombinase that allow efficient, stage-specific excision of target genes in the widely used ANKA strain.     

Expansion of experimental genetics approaches for Plasmodium berghei with versatile transfection vectors

Experimental reverse genetic approaches have proven powerful in the study of the biology of the malaria parasite. The murine malaria model parasite Plasmodium berghei is the genetically most amendable Plasmodium species and allows full access to the entire life cycle in vivo. Here, we describe a next-generation, highly versatile transfection vector set that facilitates advancing experimental genetic strategies towards a genome-wide scale. Through 36 consecutive cloning and 17 subcloning steps an optimized vector set was generated from the standard transfection plasmid. These targeting vectors, collectively referred to as the Berghei Adaptable Transfection (pBAT) plasmids, contain key elements that permit recycling of the drug-selectable cassette, robust green fluorescent labelling of recombinant parasites, carboxy-terminal tagging of target proteins with a red fluorescent-epitope tag fusion, and expression of heterologous genes. The vectors were further optimized for small size, versatile restriction endonuclease recognition sites and potential exchange of individual vector elements. We show that stable integration into a transgene expression site, an intergenic locus at a synteny breakpoint on P. berghei chromosome 6, is phenotypically silent and generated a bright green fluorescent parasite line for imaging applications. We provide an example, P. berghei actin 2, for targeted gene deletion and illustrate that the positive selection marker can be recycled, thereby permitting multiple rounds of genetic manipulations. We propose that the vectors described herein will greatly facilitate functional assignment to predicted and orphan Plasmodium gene models by multiple experimental genetics approaches.     

The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome

Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.     

von Willebrand Factor A domain-related protein, a novel microneme protein of the malaria ookinete highly conserved throughout Plasmodium parasites.

The mosquito-invasive form of the malarial parasite, the ookinete, develops numerous secretory organelles, called micronemes, in the apical cytoplasm. Micronemal proteins are thought to be secreted during midgut invasion and to play a crucial role in attachment and motility of the ookinete. We found a novel ookinete micronemal protein of rodent malarial parasite Plasmodium berghei, named P. berghei von Willebrand factor A domain-related protein (PbWARP), and report it here as a putative soluble adhesive protein of the ookinete. The PbWARP gene contained a single open reading frame encoding a putative secretory protein of 303 amino acids, with a von Willebrand factor type A module-like domain as a main component. Western blot analysis demonstrated that PbWARP was firstly produced 12 h after fertilization by maturing ookinetes as SDS-resistant complexes. Recombinant PbWARP produced with a baculovirus system also formed SDS-resistant high-order oligomers. Immuno-electron microscopic studies showed that PbWARP was randomly distributed in the micronemes. PbWARP homologues also exist in human malarial parasites, Plasmodium falciparum and Plasmodium vivax. Highly conserved primary structures of PbWARP homologues among these phylogenetically distant Plasmodium species suggest their functional significance and the presence of a common invasion mechanism widely utilized throughout Plasmodium parasites.     

Phagocyte-derived reactive oxygen species do not influence the progression of murine blood-stage malaria infections

Phagocyte-derived reactive oxygen species have been implicated in the clearance of malaria infections. We investigated the progression of five different strains of murine malaria in gp91(phox-/-) mice, which lack a functional NADPH oxidase and thus the ability to produce phagocyte-derived reactive oxygen species. We found that the absence of functional NADPH oxidase in the gene knockout mice had no effect on the parasitemia or total parasite burden in mice infected with either resolving (Plasmodium yoelii and Plasmodium chabaudi K562) or fatal (Plasmodium berghei ANKA, Plasmodium berghei K173 and Plasmodium vinckei vinckei) strains of malaria. This lack of effect was apparent in both primary and secondary infections with P. yoelii and P. chabaudi. There was also no difference in the presentation of clinical or pathological signs between the gp91(phox-/-) or wild-type strains of mice infected with malaria. Progression of P. berghei ANKA and P. berghei K173 infections was unchanged in glutathione peroxidase-1 gene knockout mice compared to their wild-type counterparts. The rates of parasitemia progression in gp91(phox-/-) mice and wild-type mice were not significantly different when they were treated with l-N(G)-methylarginine, an inhibitor of nitric oxide synthase. These results suggest that phagocyte-derived reactive oxygen species are not crucial for the clearance of malaria parasites, at least in murine models.     

Carboxypeptidases B of Anopheles gambiae as targets for a Plasmodium falciparum transmission-blocking vaccine

Anopheles gambiae is the major African vector of Plasmodium falciparum, the most deadly species of human malaria parasite and the most prevalent in Africa. Several strategies are being developed to limit the global impact of malaria via reducing transmission rates, among which are transmission-blocking vaccines (TBVs), which induce in the vertebrate host the production of antibodies that inhibit parasite development in the mosquito midgut. So far, the most promising components of a TBV are parasite-derived antigens, although targeting critical mosquito components might also successfully block development of the parasite in its vector. We previously identified A. gambiae genes whose expression was modified in P. falciparum-infected mosquitoes, including one midgut carboxypeptidase gene, cpbAg1. Here we show that P. falciparum up-regulates the expression of cpbAg1 and of a second midgut carboxypeptidase gene, cpbAg2, and that this up-regulation correlates with an increased carboxypeptidase B (CPB) activity at a time when parasites establish infection in the mosquito midgut. The addition of antibodies directed against CPBAg1 to a P. falciparum-containing blood meal inhibited CPB activity and blocked parasite development in the mosquito midgut. Furthermore, the development of the rodent parasite Plasmodium berghei was significantly reduced in mosquitoes fed on infected mice that had been immunized with recombinant CPBAg1. Lastly, mosquitoes fed on anti-CPBAg1 antibodies exhibited reduced reproductive capacity, a secondary effect of a CPB-based TBV that could likely contribute to reducing Plasmodium transmission. These results indicate that A. gambiae CPBs could constitute targets for a TBV that is based upon mosquito molecules.     

Characterisation and expression of pbs25, a sexual and sporogonic stage specific protein of Plasmodium berghei

Following gametogenesis and fertilisation in the bloodmeal within the mosquito midgut, the newly formed zygotes of the malaria parasite develop into motile invasive ookinetes. During this development, surface molecules are synthesised de novo including molecules of 21-28 kDa from the zygote-ookinete stages. An antiserum recognising a 26 kDa protein of Plasmodium berghei was used to clone the corresponding gene from a cDNA library, which was shown to be identical to the reported Pbs25 gene sequence. We show here that Pbs25 was detectable in preparations of gametes 30 min post-gametocyte activation, expression continued on zygotes, ookinetes and oocysts indicating there is a significant overlap of expression of the two immunogenic zygote-ookinete proteins belonging to the P25/28 protein family of sexual stage antigens. Biochemical analysis of Pbs25 demonstrates the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. Antibodies recognising Pbs25 impaired parasite development in the mosquito.     

Improved transfection and new selectable markers for the rodent malaria parasite Plasmodium yoelii

The rodent malaria Plasmodium yoelii is a useful model to study protective immunity to pre-erythrocytic stages of infection, pathogenesis of erythrocytic stages, and vaccine development. However, the utility of the P. yoelii model system has not been fully realized because transfection and genetic manipulation methodologies for this rodent species are less developed than that of another rodent species Plasmodium berghei. Here we report improved transfection efficiency using the AMAXA nucleofector system compared to conventional transfection methodologies. We also show that heterologous promoters from P. berghei can be used to drive expression of a green fluorescent protein (GFP) reporter protein in P. yoelii. In an effort to develop additional selectable markers for this parasite, we also tested positive selectable markers that have been used successfully in P. falciparum and P. berghei. Human dihydrofolate reductase (hdhfr) and Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (Tgdhfr-ts) conferred drug resistance to WR99210 and pyrimethamine, respectively, when introduced as episomes. These improvements should make genetic manipulation of P. yoelii more amenable and facilitate further studies of host-parasite interactions using this attractive rodent model.     

Comparative genomics in Plasmodium: a tool for the identification of genes and functional analysis.

Comparative genomics allows inferences to be drawn about the coding potential of related genomes, and the evolutionary forces that have influenced genome organisation. Early comparisons have indicated that there is significant synteny (conserved physical association of genes) between the human parasite Plasmodium falciparum and the malaria parasites of rodents, such as Plasmodium berghei. The various Plasmodium genome initiatives have now provided the opportunity to perform comparative genomics within different species of malaria parasites in more detail, allowing the discovery of orthologues and paralogues of less well conserved genes and addressing questions of conservation, evolution and structure of multi-gene families. A remarkable level of conservation is being revealed, illustrated here by a comparison of members of one of the first conserved gene families to emerge from the sequencing initiatives, the P48/45 gene family. We have identified two additional members in this family, Pf36p and Pfs38, and shown that all members are conserved in P. falciparum and P. berghei, opening the way for functional analyses in the latter more accessible rodent malaria model. In addition, it has been shown that direct comparison of a 13.6 kb contig of a chromosome of P. berghei and the orthologous region in P. falciparum reveals an unexpected high level of conservation of gene organisation and complexity. The results of this comparison highlight the value of a comparative approach to elucidate the gene content of complex loci and improve its annotation     

Evidence for major differences in ribosomal subunit proteins from Plasmodium berghei and rat liver

Purified polysomes were isolated in high yield from the erythrocytic stages of the rodent malaria parasite, Plasmodium berghei, and from rat liver. Proteins extracted from the ribosomal subunits derived from these polysomes were fractionated and their number and molecular weights were estimated by two-dimensional polyacrylamide gel electrophoresis. Plasmodial small ribosomal subunits contained 30 proteins ranging in apparent molecular size from 11.7 to 40.7 kDa, while large subunits contained 35-36 proteins ranging from 12.1 to 42.6 kDa. None of these parasite proteins was shared by the two subunits nor altered in electrophoretic mobility by radioiodination. Rat liver 40 S ribosomal subunit proteins numbered 30 and ranged from 9.2 to 37.5 kDa, while liver 60 S subunits contained 41-43 proteins with apparent molecular sizes of 10.3-45.2 kDa. Coelectrophoresis of trace amounts of radioiodinated P. berghei ribosomal subunit proteins and stainable quantities of liver proteins demonstrated that most of these 139 parasite and host ribosomal proteins possessed different two-dimensional electrophoretic mobilities under the conditions of this study. Based upon a comparative analysis of P. berghei and rodent ribosomal RNA and these data, it was concluded that parasite and host ribosomes contain distinct ribosomal RNAs and ribosomal proteins.