Ultrastructural localization of phenoloxidase in the midgut of refractory Anopheles gambiae and association of the enzyme with encapsulated Plasmodium cynomolgi

A melanogenic enzyme, phenoloxidase, was localized ultrastructurally in the midgut epithelia of 2 strains of Anopheles gambiae, a refractory strain that melanotically encapsulates Plasmodium cynomolgi ookinetes on the midgut, and a susceptible strain that does not. Midguts were incubated with either dopa or dopamine, and the resultant electron-dense product of phenoloxidase activity was localized on the basal lamina (BL) and cellular basal membrane labyrinth (BML) in uninfected mosquitoes of both strains. In infected refractory mosquitoes, the reaction products still were observed on the BL and BML but were especially dense in the BML of midgut cells near encapsulated ookinetes and in the capsule itself. In infected susceptible mosquitoes, phenoloxidase localization was reduced or absent in the BL and BML and was not observed near parasites. Phenylthiourea (PTU) inhibited the phenoloxidase reaction, indicating that the reaction product deposited in the absence of PTU resulted from enzyme activity and not autooxidation of the substrates. It is concluded that higher levels of phenoloxidase in the refractory strain following a blood meal may contribute to the ability to encapsulate ookinetes.     

A study on pathogenicity and mosquito transmission success in the rodent malaria parasite Plasmodium chabaudi adami

We investigated the parasitology, pathogenicity (virulence) and infectivity to mosquitoes of blood infections in mice, of two strains, DS and DK, of the rodent malaria parasite Plasmodium chabaudi adami. Blood infections of DS were found to be highly pathogenic; the asexual parasites in these infections were fast-growing and showed no evidence of selectivity in their infection of host erythrocytes. In contrast to DS, blood infections of DK were much less pathogenic; the asexual parasites were slower-growing and showed a moderate degree of selectivity to a subset of erythrocytes which were not reticulocytes. In both DS and DK infections, infectivity to mosquitoes was highest before the peak of asexual parasitaemia had occurred; usually this did not coincide with the time when gametocyte numbers in the blood were highest. Infections with the pathogenic DS strain in CBA mice produced fewer gametocytes than did the less pathogenic DK strain. The DS strain infections in both CBA and C57 mice were also significantly much less infective to mosquitoes than the DK strain. Investigations by others on the related rodent malaria parasite subspecies, Plasmodium chabaudi chabaudi, have indicated that the mosquito infectivity of blood infections in mice tended to be higher in the more pathogenic (virulent) and lower in the less pathogenic strains of this parasite subspecies. This is the converse of the finding of the present investigation of blood infections of P. c. adami in mice in which a more pathogenic, or virulent, strain (DS) of these parasites was significantly much less infective to mosquitoes than was a less pathogenic strain (DK).     

Isolation,characterization and function of laccase from Trichoderma

Of fourteen natural isolates of Trichoderma, no correlation was found between substrate weight loss and phenol oxidase (PO) activity in rice straw cultures. The highest PO producer from these laccase-positive strains was subjected to UV mutagenesis in order to select high and low PO activity mutants. There was no significant difference in substrate weight loss for mutant strains with six times higher and six times lower PO activity than the parent strain. Nor did the enzyme activity result in decreased growth inhibition by inhibitory phenolic compounds. PO enzyme from the parent Trichoderma and one of its high-PO-activity mutants was subsequently purified by ethanol precipitation from liquid cultures optimized by supplementation with copper sulphate and cycloheximide. Protein staining and activity staining of disc electrophoresis gels showed that only one PO enzyme of approximately 71 000 Da was produced. The enzyme could be defined as a laccase (benzenediol: oxygen oxidoreductase E.C. 1.10.3.2) because it catalysed the oxidation of syringaldazine and p-phenylenediamine in the absence of hydrogen peroxide, and because it was inhibited by cetyltrimethylammonium because but not by cinnamic acid. No specific in-vivo function could be assigned to this enzyme.Correspondence to: T. W. Flegel     

Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants

We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl-N'-nitro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.     

Metacytofilin has potent anti-malarial activity

Metacytofilin (MCF) was isolated from the fungus Metarhizium sp. TA2759. Although MCF possesses anti-Toxoplasma activity, the effects of this compound against other parasites are unknown. Here, we evaluated the in vitro anti-malarial activity of MCF against the 3D7 strain and the chloroquine-resistant K1 strain of Plasmodium falciparum. The half maximal inhibitory concentrations (IC₅₀) of MCF against the 3D7 and K-1 strains following culture for 48 h were 666 nM and 605 nM, respectively. Artemisinin was more potent than MCF against both strains (3D7 IC₅₀: 17.4 nM; K-1 IC₅₀: 18.3 nM), while chloroquine was ineffective against the chloroquine-resistant strain (3D7 IC₅₀: 39.1 nM; K-1 IC₅₀: 1.62 μM). MCF affected the ring stage of the parasites, resulting in their death as shown by spots within red blood cells. MCF also inhibited parasite growth following culture for 72 h (3D7 IC₅₀, 285 nM). Four optical isomers of cyclo[Leu-Phe]-diketopiperazine derivatives with modified methoxy and/or hydroxyl groups lost anti-malarial activity, suggesting that the spatial positions of the methoxy and hydroxyl groups in MCF play an important role in its anti-malarial effects. Together, these data suggest that MCF may represent a promising lead compound for treatment of drug-resistant malarial parasites.     

β-Galactosidase of Propionibacterium shermanii

Ten strains of Propionibacterium shermanii were tested for beta-galactosidase (beta-gal) activity. Of these ten strains, five yielded enhanced enzyme activity when cell suspensions were treated with toluene-acetone; on solvent treatment, the remaining five lost a considerable portion of the activity found in whole-cell suspensions. By using a strain yielding decreased activity upon solvent treatment, explanations for the loss in activity were sought through assays for possible alternative beta-galactoside utilization mechanisms. When this strain was assayed for beta-D-phosphogalactoside galactohydrolase by using orthonitrophenyl-beta-D-galactopyranoside-6-P04 as a substrate, the activity was wither lower or indiffernt as compared with beta-gal activity determined simultaneously. Cell suspensions of P. shermanii 7 and 22 (strains chosen for further work) grown separately on the individual substrates (lactose, glucose, galactose, and sodium lactate) did not show significant differences in beta-gal activity. Optimal temperature for beta-gal activity in untreated and toluene-acetone-treated cell suspensions of strain 7 was 52 C. With strain 22, of the temperatures tested, maximal activity in untreated cell suspensions was noted at 58 C and with solvent-treated cells at 32 C. In the cell-free extract (CFE) system, both strains exhibited maximal activity at 52 C. Optimal pH for untreated and solvent-treated cell suspensions of both strains was around 7.5. In the P. shermanii 22 CFE system, maximal activity occurred at pH 7.0; pH had very little effect on enzyme activity in P. shermanii 7 CFE. Sodium or potassium phosphate buffers in the assay system yielded the best activity. In the CFE system of these two strains, Mn2+ was definitely stimulatory, but in untreated and solvent-treated cell systems of these strains presence or absence of Mn2+ in the assay system had variable effects on enzyme activity. Maximal beta-gal activity was noted in P. shermanii 7 cells harvested after 28 h of growth at 32 C in sodium lactate broth. Sulfhydryl-group blocking agents inhibited enzyme activity in P. shermanii 22 CFE; the inhibition was partly reversed by dithiothreitol.     

Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei

The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite.     

Characterization of cross-reactive blood-stage antigens of the Plasmodium cynomolgi complex using anti-Plasmodium vivax monoclonal antibodies

Five out of 18 monoclonal antibodies (moAB's) produced against blood stages of a Brazilian (Belem) strain of Plasmodium vivax were shown to cross-react with all of the 11 strains of the P. cynomolgi complex that were assayed. The 5 moAB's produced 3 different patterns of immunofluorescence, identical for both P. vivax and P. cynomolgi. Three of these moAB's appeared to react with antigens associated with the cytoplasm or membranes of infected erythrocytes. By Western blot analysis, 2 of these 3 moAB's identified an antigen with an apparent molecular weight of 31 kDa in extracts of parasitized erythrocytes of both species; the third of these moAB's reacted with an antigen with an apparent molecular weight of 95 kDa. By immunofluorescence, the 2 other moAB's reacted only with parasites at all developmental stages. The target antigen of these 2 moAB's was not identified. Immunoradiometric assays indicated that the moAB's are directed against 3 or possibly 4 distinct nonrepetitive epitopes. None of the moAB's inhibited merozoite invasion or growth of the parasites in an in vitro culture system of the Berok strain of P. cynomolgi.     

Variation among Plasmodium falciparum strains in their reliance on mitochondrial electron transport chain function

Previous studies demonstrated that Plasmodium falciparum strain D10 became highly resistant to the mitochondrial electron transport chain (mtETC) inhibitor atovaquone when the mtETC was decoupled from the pyrimidine biosynthesis pathway by expressing the fumarate-dependent (ubiquinone-independent) yeast dihydroorotate dehydrogenase (yDHODH) in parasites. To investigate the requirement for decoupled mtETC activity in P. falciparum with different genetic backgrounds, we integrated a single copy of the yDHODH gene into the genomes of D10attB, 3D7attB, Dd2attB, and HB3attB strains of the parasite. The yDHODH gene was equally expressed in all of the transgenic lines. All four yDHODH transgenic lines showed strong resistance to atovaquone in standard short-term growth inhibition assays. During longer term growth with atovaquone, D10attB-yDHODH and 3D7attB-yDHODH parasites remained fully resistant, but Dd2attB-yDHODH and HB3attB-yDHODH parasites lost their tolerance to the drug after 3 to 4 days of exposure. No differences were found, however, in growth responses among all of these strains to the Plasmodium-specific DHODH inhibitor DSM1 in either short- or long-term exposures. Thus, DSM1 works well as a selective agent in all parasite lines transfected with the yDHODH gene, whereas atovaquone works for some lines. We found that the ubiquinone analog decylubiquinone substantially reversed the atovaquone inhibition of Dd2attB-yDHODH and HB3attB-yDHODH transgenic parasites during extended growth. Thus, we conclude that there are strain-specific differences in the requirement for mtETC activity among P. falciparum strains, suggesting that, in erythrocytic stages of the parasite, ubiquinone-dependent dehydrogenase activities other than those of DHODH are dispensable in some strains but are essential in others.     

Changes in schizogony and drug response in two lines of rodent Plasmodium, P. berghei NK 65 and P. berghei ANKA

White mice were infected with two strains, ANKA and NK 65, of Plasmodium berghei. The parasites were subjected to chloroquine pressure (60 mg/kg at each passage) during 20 passages. We then compared the behaviour of the strains as they acquired chemoresistance. The drug resistance was estimated by the 2% delay time test (D2%), and the schizogonic rhythm by the synchronicity index (SI). Before drug pressure, the ANKA strain had a D2% of 4.34, and a SI of 0.2. This strain became highly drug resistant, but synchronicity increased: the D2% was 2.93, and the SI was 0.36 at the 20th passage. The NK 65 strain had an initial D2% of 4.12, and an SI of 0.2. The chemoresistance acquired during 20 passages was very irregular for this strain: after drug pressure, the D2% was 2.03 and the SI was 0.28. Drug pressure was then removed (for both strains), for 10 passages (no chloroquine). Resistance and synchronicity returned to their initial values. The two strains behaved very differently, in terms of their affinity for reticulocytes, and with chloroquine activity which favours an increase in SI because only merozoites are preserved.     

Attenuated Plasmodium yoelii lacking purine nucleoside phosphorylase confer protective immunity

Malaria continues to devastate sub-Saharan Africa owing to the emergence of drug resistance to established antimalarials and to the lack of an efficacious vaccine. Plasmodium species have a unique streamlined purine pathway in which the dual specificity enzyme purine nucleoside phosphorylase (PNP) functions in both purine recycling and purine salvage. To evaluate the importance of PNP in an in vivo model of malaria, we disrupted PyPNP, the gene encoding PNP in the lethal Plasmodium yoelii YM strain. P. yoelii parasites lacking PNP were attenuated and cleared in mice. Although able to form gametocytes, PNP-deficient parasites did not form oocysts in mosquito midguts and were not transmitted from mosquitoes to mice. Mice given PNP-deficient parasites were immune to subsequent challenge to a lethal inoculum of P. yoelii YM and to challenge from P. yoelii 17XNL, another strain. These in vivo studies with PNP-deficient parasites support purine salvage as a target for antimalarials. They also suggest a strategy for the development of attenuated nontransmissible metabolic mutants as blood-stage malaria vaccine strains.     

Novel squaramides with in vitro liver stage antiplasmodial activity

A structure–activity relationship study was performed with ten 8-aminoquinoline-squaramides compounds active against liver stage malaria parasites, using human hepatoma cells (Huh7) infected by Plasmodium berghei parasites. In addition, their blood-schizontocidal activity was assessed against chloroquine-resistant W2 strain Plasmodium falciparum. Compound 3 was 7.3-fold more potent than the positive control primaquine against liver-stage parasites, illustrating the importance of the squarate moiety to activity.     

Exacerbation of experimental Trypanosoma cruzi infection in mice by concomitant malaria.

The effect of malaria on the chronic phase of Chagas' disease was investigated in mice. The animals were given Plasmodium bergheri-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y. CL and Gilmar). in all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas' disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curved of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phas with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Strain-Specific Protective Effect of the Immunity Induced by Live Malarial Sporozoites under Chloroquine Cover

The efficacy of a whole-sporozoite malaria vaccine would partly be determined by the strain-specificity of the protective responses against malarial sporozoites and liver-stage parasites. Evidence from previous reports were inconsistent, where some studies have shown that the protective immunity induced by irradiated or live sporozoites in rodents or humans were cross-protective and in others strain-specific. In the present work, we have studied the strain-specificity of live sporozoite-induced immunity using two genetically and immunologically different strains of Plasmodium cynomolgi, Pc746 and PcCeylon, in toque monkeys. Two groups of monkeys were immunized against live sporozoites of either the Pc746 (n = 5), or the PcCeylon (n = 4) strain, by the bites of 2–4 sporozoite-infected Anopheles tessellates mosquitoes per monkey under concurrent treatments with chloroquine and primaquine to abrogate detectable blood infections. Subsequently, a group of non-immunized monkeys (n = 4), and the two groups of immunized monkeys were challenged with a mixture of sporozoites of the two strains by the bites of 2–5 infective mosquitoes from each strain per monkey. In order to determine the strain-specificity of the protective immunity, the proportions of parasites of the two strains in the challenge infections were quantified using an allele quantification assay, Pyrosequencing™, based on a single nucleotide polymorphism (SNP) in the parasites’ circumsporozoite protein gene. The Pyrosequencing™ data showed that a significant reduction of parasites of the immunizing strain in each group of strain-specifically immunized monkeys had occurred, indicating a stronger killing effect on parasites of the immunizing strain. Thus, the protective immunity developed following a single, live sporozoite/chloroquine immunization, acted specifically against the immunizing strain and was, therefore, strain-specific. As our experiment does not allow us to determine the parasite stage at which the strain-specific protective immunity is directed, it is possible that the target of this immunity could be either the pre-erythrocytic stage, or the blood-stage, or both.     

Susceptibility of five strains of mice to Babesia microti of human origin.

One outbred (CF1) and four inbred (BALB/c, C57, CBA and C3H) strains of mice were tested for susceptibility to Babesia microti of human origin. Of these, intact C3H mice developed higher parasitemia than all other intact mice, while BALB/c mice developed the highest parasitemia among splenectomized mice. Susceptibility was not related to H-2 haplotype in any obvious way. Because C3H and BALB/c mice developed relatively high initial peak parasitemias, the parasite was serially passaged in both of these mouse strains in an attempt to increase parasite virulence. After 30 passages in BALB/c and 49 passages in C3H mice over a period of 12 months, maximum parasitemias were 50 times higher than those observed initially. After the peak parasitemias of these two mouse-adapted parasites had stabilized, the relationship between onset and level of maximum parasitemia and number of parasites inoculated was determined. With both C3H- and BALB/c-adapted parasites, as inoculum size increased, the time required to reach maximum parasitemia decreased and the level of maximum parasitemia increased. Studies involving infection of either mouse strain with parasites adapted to the heterologous mouse strain indicated that C3H mice were more susceptible than BALB/c mice to homologous or heterologous parasites. These data suggest that the virulence of B. microti to the mouse can be increased by prolonged passage in this host. Once adaptation to this host species has occurred, virulence appears to be more dependent on the innate susceptibility of the mouse strain than on adaptation of the parasites to a particular strain of mouse.     

Plasmodium falciparum purine nucleoside phosphorylase is critical for viability of malaria parasites

Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (Deltapfpnp). Lysates of the Deltapfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the Deltapfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and Deltapfpnp parasites. The Deltapfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.     

Enzymatic antioxidant response to low-temperature acclimation in the cyanobacterium <Emphasis Type="Italic">Arthrospira platensis</Emphasis>

Changes in antioxidant enzyme activities in response to low-temperature-induced photoinhibition were investigated in the two strains of the cyanobacterium Arthrospira platensis, Kenya and M2. When transferred to 15°C from 33°C, cells exhibited an immediate cessation of growth followed by a new acclimated growth rate. Although both strains had similar growth rates at 33°C, once transferred to a lower temperature environment, Kenya had a faster growth rate than M2. There were variations in the antioxidant enzyme activities of both strains during 15°C acclimation. The activity of superoxide dismutase from Kenya was higher than that from M2 and increased remarkably with acclimation time. Catalase activity of both strains increased at first but decreased later in the acclimation process. Ascorbate-dependent peroxidase activity of the Kenya strain declined when transferred to the low-temperature environment while peroxidase activity of M2 decreased in the beginning and then increased with time. The dehydroascorbate reductase activity of both strains was variable during the acclimation period while the glutathione reductase activity was not modified immediately. Our finding may support that the faster growth rate of the Kenya strain at lower temperatures as compared with the M2 strain might be explained by the higher antioxidant enzyme activities of Kenya at lower temperatures and through its ability to apply a more efficient regulatory strategy of enzymatic antioxidant response to low-temperature-induced photoinhibition.