Mitochondrial RNA polymerase is an essential enzyme in erythrocytic stages of Plasmodium falciparum

Apicomplexan parasites are serious pathogens of animals and man that cause diseases including coccidiosis, malaria and toxoplasmosis. The importance of these parasites has prompted the establishment of genomic resources in support of developing effective control strategies. For the Eimeria species resources have developed most rapidly for the reference Eimeria tenella Houghton strain (http://www.genedb.org/Homepage/Etenella). The value of these resources can be enhanced by comparison with related parasites. The well characterised immunogenicity and genetic diversity associated with Eimeria maxima promote its use in genetics-led studies on coccidiosis and recommended its selection for sequencing. Using a combination of sequencing technologies a first draft assembly and annotation has been produced for an E. maxima Houghton strain-derived clone (EmaxDB; http://www.genomemalaysia.gov.my/emaxdb/). The assembly of a draft genome sequence for E. maxima provides a resource for comparative studies with Eimeria and related parasites as demonstrated here through the identification of genes predicted to encode microneme proteins in E. maxima.     

Antisense oligonucleotides targeting malarial aldolase inhibit the asexual erythrocytic stages of Plasmodium falciparum

A major obstacle in the global effort to control malaria is the paucity of anti-malarial drugs. This is compounded by the continuing emergence and spread of resistance to old and new anti-malarial drugs in the malarial parasites. Here we describe the anti-malarial effect of phosphorothioate antisense (AS) oligodeoxynucleotides (ODNs) targeting the aldolase enzyme of Plasmodium falciparum, using the asexual blood stages of the parasite grown in vitro. The blood stages of P. falciparum depend almost entirely on the energy produced by their own glycolysis. Aldolase, the fourth enzyme of the glycolytic pathway, is highly upregulated during the malarial 48-h life cycle. We found that the mRNA of this enzyme can be inhibited, in a sequence specific manner, using AS-ODN to the splice sites on the pre-mRNA of malarial aldolase. At the enzyme level, both specific AS-ODNs for the splice sites, as well as for the translation initiation site on mature mRNA, can inhibit aldolase enzyme activity within the trophozoites of P. falciparum. Furthermore, this downregulation of the malarial aldolase results in a reduction in the production of ATP within the parasite. Finally, the treatment reduces parasitemia. In summary, AS-ODNs targeting the aldolase gene of P. falciparum can interfere with the blood-stage life cycle of this parasite in vitro by inhibiting the expression of the enzyme aldolase which results in decreased malarial glycolysis and energy production. Thus, we conclude that blockade of the expression of malarial glycolytic enzymes using specific AS-ODNs has the potential of a new anti-malarial strategy.     

Experimental infection of nude mice as a model for Sarcocystisneurona-associated encephalitis

The development of a rodent model for the study of Sarcocystisneurona encephalitis is described. Animal models have been developed for a number of protozoal parasites; however, no such model exists for S. neurona. The approach used in this study is similar to that employed for other closely related protozoal parasites such as Neosporacaninum and Toxoplasmagondii. A time course of infection was examined, and histopathology, immunohistochemistry, and parasite isolation were used to examine the pathogenesis and follow the infection from 1 to 6 weeks postinoculation. S.neurona was associated with the development of encephalitis in these mice, and the immune status determined the susceptibility of these mice to S. neurona-associated encephalitis. Received: 11 February 1997 / Accepted: 24 April 1997     

Magnetic separation: a highly effective method for synchronization of cultured erythrocytic Plasmodium falciparum

The magnetic method has been previously utilized to concentrate Plasmodium-infected erythrocytes without any significant influence on the viability of the parasite. We attempted, in this study, to concentrate and synchronize cultivated P. falciparum via the magnetic method. The results of this study showed that the magnetic method effectively synchronized and concentrated P. falciparum with finer demarcation capacity in the erythrocytic asexual cycle of the parasite than currently available synchronization methods. Concentration and synchronization by the magnetic method proved most effective when schizonts were dominant. Therefore, it proved necessary to enhance the synchronization efficiency of the magnetic method by first applying the method currently in use, which renders schizonts dominant. Our study also showed that the intrinsic life cycle of erythrocytic P. falciparum was slightly longer than 48 h observed in natural infection cases, and that the length of the intrinsic life cycles between various P. falciparum strains differed slightly. Sun-Young Ahn and Mi-Young Shin contributed equally to this work.     

Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection

Genetic manipulation of Plasmodium falciparum in culture through transfection has provided numerous insights into the molecular and cell biology of this parasite. The procedure is rather cumbersome, and is limited by the number of drug-resistant markers that can be used for selecting transfected parasites. Here we report a new selectable marker that could allow multiple transfections. We have taken advantage of our finding that a critical function of the mitochondrial electron transport chain (mtETC) in the erythrocytic stages of P. falciparum is the regeneration of ubiquinone as co-substrate of dihydroorotate dehydrogenase (DHODH), and that transgenic P. falciparum expressing ubiquinone-independent DHODH from yeast (yDHODH) are resistant to all mtETC inhibitors. We assessed the possibility of using yDHODH as a positive selectable marker for transfections of P. falciparum, including its use in gene disruption strategies. We constructed a transfection vector designed for gene disruption, termed pUF-1, containing the yDHODH gene as the positive selection marker in combination with a previously described fused yeast cytosine deaminase-uracil phosphoribosyl transferase gene as a negative selection marker. Transfection of the D10 strain followed by selection with atovaquone yielded positively selected parasites containing the plasmid, demonstrating that yDHODH can be used as a selective marker. Atovaquone, however, could not be used for such selection with the Dd2 strain of P. falciparum. On the other hand, we demonstrated that yDHODH transgenic parasites could be selected in both strains by Plasmodium DHODH-specific triazolopyrimidine-based inhibitors. Thus, selection with DHODH inhibitors was superior in that it successfully selected transgenic Dd2 parasites, as well as yielded transgenic parasites after a shorter period of selection. As a proof of concept, we have successfully disrupted the type II vacuolar proton-pumping pyrophosphatase gene (PfVP2) in P. falciparum by double crossover recombination, showing that this gene is not essential for the survival of blood stage parasites.     

Obtaining hepatic stages of Plasmodium falciparum in vitro

Sporozoites of Plasmodium falciparum, obtained by membrane feeding of Anopheles freeborni or A. stephensi with cultured gametocytes, were used to infect monolayers of human hepatocytes. Fluorescent labelling with an African serum as well as Giemsa staining performed from day one to day 7 of cultures, demonstrated the presence of numerous hepatic schizonts measuring up to 40 micron.     

Experimental erythrocytic malaria infection induces elevated serum amyloid P production in mice

Experimental blood-stage malaria infection of NIH mice was observed to induce an acute phase response (APR). Infection of mice with either P. chabaudi, P. vinckei (both non-lethal) or P. berghei (lethal infection) resulted in elevated serum amyloid P (SAP) production, the major acute phase protein in mice. Peak production occurred at the peak of the parasitaemia (approximately day 10 post infection). SAP isolated from the serum of P. chabaudi infected mice was shown to inhibit the growth of intra-erythrocytic malaria parasites in vitro. Furthermore, isolated SAP suppressed the proliferative response of splenocytes taken from a na?ve mouse to concanavalin A. To assess if SAP had a protective role in vivo during experimental blood-stage infection, IL-6 deficient mice, which have a significantly reduced APR, were infected with P. chabaudi. A significant extension to the primary parasitaemia was observed in IL-6 deficient mice compared to infected wild type mice. These observations demonstrate that blood-stage malaria infection induces a systemic APR and that this may contribute to the immune response to infection in an anti-parasitic or immunomodulatory manner.     

Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum

The limited number of selectable markers available for malaria transfection has hindered extensive manipulation of the Plasmodium falciparum genome and subsequently thorough genetic analysis of this organism. In this paper, we demonstrate that P. falciparum is highly sensitive to the drug puromycin, but that transgenic expression of the puromycin-N-acetyltransferase (PAC) gene from Streptomyces alboninger confers resistance to this drug with the IC(50) and IC(90) values increasing approximately 3- and 7-fold, respectively in PAC-expressing parasites. Despite this relatively low level of resistance, parasite populations transfected with the PAC selectable marker and selected directly on puromycin emerged at the same rate post-transfection as human dihydrofolate reductase (hDHFR)-expressing parasites, selected independently with the anti-folate drug WR99210. Transfected parasites generally maintained the PAC expression plasmid episomally at between two and six copies per parasite. We also demonstrate by cycling transfected parasites in the presence and absence of puromycin for several weeks, that the PAC selectable marker can be used for gene-targeting. Since the mode of action of puromycin is distinct from other drugs currently used for the stable transfection of P. falciparum, the PAC selectable marker should also have applicability for use in conjunction with other positive selectable markers, thereby increasing the possibilities for more complex functional studies of this organism.     

The A/T-specific DNA alkylating agent adozelesin inhibits Plasmodium falciparum growth in vitro and protects mice against Plasmodium chabaudi adami infection

There is an urgent need for new anti-malarial drugs to combat the resurgence of resistance to current therapies. To exploit the A/T richness of malaria DNA as a potential target for anti-malarial drugs we tested an A/T-specific DNA synthesis inhibitor, adozelesin, for activity against Plasmodium falciparum in vitro and Plasmodium chabaudi adami in mice. Adozelesin is a DNA alkylating agent that exhibits specificity for the motif A/T, A/T and A. In P. falciparum 3D7 cultures, adozelesin acts as a powerful inhibitor of parasite growth (IC(50) of 70 pM) and is equally potent at killing the drug-resistant strains FCR3 and 7G8. Using a real-time PCR assay, we show that treatment with adozelesin in vitro results in damage of P. falciparum genomic DNA. In synchronized cultures, adozelesin exhibits a concentration-dependent effect on parasitemia and on the development of parasites through the asexual cycle. In asynchronous cultures, parasites arrest at all stages of the asexual cycle suggesting that adozelesin exerts other anti-parasitic effects in addition to inhibiting DNA replication. These anti-parasite effects are irreversible since cultures exposed to adozelesin for more than 6h fail to recover upon removal of the drug. Furthermore, adozelesin is very effective at suppressing malaria infection in vivo; growth of P. c. adami DK in mice was highly impaired by a single injection of adozelesin (25 microg/kg) at 4 days post-infection. These results demonstrate that adozelesin irreversibly blocks parasite growth in vitro and suppresses parasite infection in vivo, suggesting that A/T-specific DNA damaging agents represent a new class of compounds with potential as anti-malarials.     

Experimental meningococcal infection in mice: a model for mucosal invasion.

A more complete understanding of meningococcal disease has been hampered by the lack of an appropriate animal model. Previous models have utilized injections of meningococci, which precludes the study of nasopharyngeal colonization and invasion. We have developed a model for meningococcal disease in which litters of 5-day-old mice are challenged intranasally with 10(7) viable meningococci. Bacteremia is monitored by jugular venous blood cultures, and cerebrospinal fluid is sampled by cisternal punctures. Human disease-associated and carrier strains were compared; nasopharyngeal colonization was similar for these bacteria, but the case-associated strains were much more frequently invasive and caused bacteremia. Twenty-one percent of bacteremic animals had meningitis. There was an age-related susceptibility to infection which correlated inversely with the levels of serum complement. Preinjection of iron dextran increased the number of animals which were bacteremic, the concentration of bacteria in blood, and nasopharyngeal colonization. Noncapsular variants of virulent meningococci did not colonize nasopharyngeal tissue in vivo, and they were not invasive. This neonatal mouse model mimics meningococcal disease as seen in humans and may be useful in studying the initial events in the pathogenesis of meningococcal disease.     

恶性疟复合多价抗原基因在小鼠中免疫应答的初步研究

目的探索恶性疟复合多价ＤＮＡ疫苗的可行性。方法把带有ＡＴＧ的接头与人工合成的恶性疟原虫复合多价抗原基因ＡＢ相连后，构建分别带有ＳＶ４０或ＲＳＶ启动子的真核表达载体ｐＳＶ２／ＡＢ及ｐＲＥＰ９／ＡＢ，重组表达质粒经肌肉注射免疫ＢＡＬＢ／ｃ小鼠后，检测其诱发特异性体液和细胞免疫应答水平及毒副作用。结果ｐＳＶ２／ＡＢ及ｐＲＥＰ９／ＡＢ免疫ＢＡＬＢ／ｃ小鼠后均诱发了一定水平的细胞及体液免疫应答，带ＲＳＶ启动子的ｐＲＥＰ９／ＡＢ免疫原性略强于带ＳＶ４０启动子的ｐＳＶ２／ＡＢ，ＤＮＡ免疫后未见明显的毒副作用。结论恶性疟复合多价ＤＮＡ疫苗可诱发特异的免疫应答，为疟疾ＤＮＡ疫苗的研究提供了一定的理论及实验依据     

Transgenic mice expressing human transferrin as a model for meningococcal infection

The pathogenesis of meningococcal disease is poorly understood due to the lack of a relevant animal model. Moreover, the use of animal models is not optimal as most meningococcal virulence determinants recognize receptors that are specifically expressed in human tissues. One major element of the host specificity is the system of meningococcal iron uptake by transferrin-binding proteins that bind specifically human transferrin but not murine transferrin. We developed a new mouse model for experimental meningococcal infection using transgenic mice expressing human transferrin. Intraperitoneal challenge of transgenic mice induced bacteremia for at least 48 h with an early stage of multiplication, whereas the initial inoculum was rapidly cleared from blood in wild-type mice. Inflammation in the subarachnoidal space with a high influx of polymorphonuclear cells was observed only in transgenic mice. Meningococcal mutants that were unable to use transferrin as a source of iron were rapidly cleared from both wild-type and transgenic mice. Thus, transgenic mice expressing human transferrin may represent an important advance as a new mouse model for in vivo studies of meningococcal virulence and immunogenicity factors.     

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.     

Evaluation & usefulness of a immunochromatographic test for rapid detection of Plasmodium falciparum infection

Rapid test (Parachek Pf) based on detection of HRP-2 protein specific to Plasmodium falciparum by immunochromatographic technique was evaluated. Prevalence of infection was 8.5%. The test was 100% sensitive & 99.5% specific on comparison with light microscopy. The test is useful for making 'on the spot' diagnosis.     

Neutral lipid synthesis and storage in the intraerythrocytic stages of Plasmodium falciparum

In eukaryotic cells the neutral lipids, steryl esters and triacylglycerol, are synthesized by membrane-bound O-acyltransferases and stored in cytosolic lipid bodies. We show here that the intraerythrocytic stages of Plasmodium falciparum produce triacylglycerol using oleate and diacylglycerol as substrates. Parasite membrane preparations reveal a synthesis rate of 4.5 +/- 0.8 pmol x min(-1)mg(-1) of protein with maximal production occurring in the mid- and late-trophozoite stages in both, membrane preparations and live parasites. In contrast to other eukaryotic cells, no discernable amounts of steryl esters are produced, and the parasite is insensitive to cholesterol esterification inhibitors. Synthesized neutral lipids are stored as lipid bodies in the parasite cytosol in a stage specific manner. Their biogenesis is not modified upon incubation with excess fatty acids or lipoproteins or after lipoprotein depletion of the culture medium. We investigated on the enzymes involved in neutral lipid synthesis and found that only one gene with significant homology to known members of the membrane-bound O-acyltransferase family is present in the P. falciparum genome. It encodes a microsomal transmembrane protein with a predicted size of 78.1 kDa, which we named PfDGAT because of its close identity with various known acyl-CoA:diacylglycerol acyltransferases. PfDGAT is expressed in a stage specific manner as documented by Western blotting and immunoprecipitation assays using antibodies against Toxoplasma DGAT, suggesting that PfDGAT is the most likely candidate for plasmodial triacylglycerol synthesis.