Enhanced Uptake and Metabolism of Riboflavin in Erythrocytes Infected with Plasmodium falciparum

Riboflavin deficiency inhibits the growth of malaria parasites both in vitro and in vivo in infected animals and humans. Although the precise mechanisms underlying this inhibition are unknown, they may involve enhanced requirements for riboflavin by parasites. To investigate this possibility, the rate of uptake of [¹⁴C]riboflavin and the biosynthesis of FMN and FAD from riboflavin were studied in infected (5–8% parasitemia) and uninfected human erythrocytes. All cells were incubated for 0–3 h at 37° C in phosphate buffered saline containing MgCl₂, glucose, and [¹⁴C]riboflavin (2.5–7.5 μM). At hourly intervals, samples were removed, centrifuged, washed twice with cold buffer, and lysed before counting the radioactivity. The rate of in vitro biosynthesis of FMN and FAD from riboflavin in erythrocytes was measured by ion exchange chromatography and reverse isotope dilution techniques. Results showed that the rate of riboflavin uptake and the biosynthesis of FMN and FAD were enhanced in erythrocytes with parasitemia as compared with results in unparasitized erythrocytes. Riboflavin uptake in erythrocytes was proportional to the extent of parasitemia and especially to percent of schizonts present in erythrocytes. These studies indicate that the requirement for riboflavin may be greater in the parasite than in the host erythrocyte. This increased riboflavin requirement may be due to rapid multiplication, higher metabolic rate, and extreme vulnerability to oxidative stress of malaria parasites compared with that of host erythrocytes. The differential requirement of riboflavin by the host and the malaria parasite may hold important potential for developing new strategies for malaria chemotherapy.     

New Export Pathway in Plasmodium falciparum‐Infected Erythrocytes: Role of the Parasite Group II Chaperonin,PfTRiC

The export of numerous proteins to the plasma membrane of its host erythrocyte is essential for the virulence and survival of the malaria parasite Plasmodium falciparum. The Maurer's clefts, membrane structures transposed by the parasite in the cytoplasm of its host erythrocyte, play the role of a marshal platform for such exported parasite proteins. We identify here the export pathway of three resident proteins of the Maurer's clefts membrane: the proteins are exported as soluble forms in the red cell cytoplasm to the Maurer's clefts membrane in association with the parasite group II chaperonin (PfTRIC), a chaperone complex known to bind and address a large spectrum of unfolded proteins to their final location. We have also located the domain of interaction with PfTRiC within the amino‐terminal domain of one of these Maurer's cleft proteins, PfSBP1. Because several Maurer's cleft membrane proteins with different export motifs seem to follow the same route, we propose a general role for PfTRiC in the trafficking of malarial parasite proteins to the host erythrocyte.      

Detection and cartography of the fluorinated antimalarial drug mefloquine in normal and Plasmodium falciparum infected red blood cells by scanning ion microscopy and mass spectrometry

Summary— Due to the presence of fluorine atoms in its molecule, the antimalarial drug mefloquine (MQ) can be easily detected in normal and Plasmodium falciparum infected red blood cells (RBC) by scanning ion microscopy and mass spectrometry. The P falciparum infected RBC exhibited intense distribution of MQ inside the parasite. The main compartments of the parasite which accumulate the drug were the food vacuole and the cytoplasm. The correlation between fluorine (¹⁹F^?) and phosphorus (³¹P^?) as well as probes for the DNA synthesis (BrdU and IdU) emissions shows that the parasite nucleus is also accessible to the drug. This study demonstrates that SIMS technique on smear preparations is an efficient approach for the direct detection and cartography of fluorinated antimalarial drugs in normal and P falciparum infected RBC, without radioactive labelling.     

Metabolic changes accompanying the loss of fumarate hydratase and malate–quinone oxidoreductase in the asexual blood stage of Plasmodium falciparum

In the glucose-rich milieu of red blood cells, asexually replicating malarial parasites mainly rely on glycolysis for ATP production, with limited carbon flux through the mitochondrial tricarboxylic acid (TCA) cycle. By contrast, gametocytes and mosquito-stage parasites exhibit an increased dependence on the TCA cycle and oxidative phosphorylation for more economical energy generation. Prior genetic studies supported these stage-specific metabolic preferences by revealing that six of eight TCA cycle enzymes are completely dispensable during the asexual blood stages of Plasmodium falciparum, with only fumarate hydratase (FH) and malate–quinone oxidoreductase (MQO) being refractory to deletion. Several hypotheses have been put forth to explain the possible essentiality of FH and MQO, including their participation in a malate shuttle between the mitochondrial matrix and the cytosol. However, using newer genetic techniques like CRISPR and dimerizable Cre, we were able to generate deletion strains of FH and MQO in P. falciparum. We employed metabolomic analyses to characterize a double knockout mutant of FH and MQO (ΔFM) and identified changes in purine salvage and urea cycle metabolism that may help to limit fumarate accumulation. Correspondingly, we found that the ΔFM mutant was more sensitive to exogenous fumarate, which is known to cause toxicity by modifying and inactivating proteins and metabolites. Overall, our data indicate that P. falciparum is able to adequately compensate for the loss of FH and MQO, rendering them unsuitable targets for drug development.     

生物正交化学在活体标记及药物传递中的研究进展

生物正交化学反应是一类可以在生理条件下发生的化学反应,具有简单、高效、高特异性的特点,在生物医学的研究中被广泛应用.基于生物体天然生命过程的代谢工程,可对生物分子进行无损、高效的生物代谢修饰,是一种理想的生物修饰技术.通过生物代谢途径可有效地将各种化学报告基团引入靶标物的生物分子中,有利于携带配对基团的标记物与其发生生物正交反应,从而在活体系统中实现生物分子的标记示踪和药物递送.这种基于代谢工程与生物正交化学的标记策略因为具有两者之间的优势,在生物医学工程中的标记、成像示踪、诊断等领域展现出巨大的研究价值与应用潜力.本文介绍了生物正交和代谢工程的原理与生物医学研究进展,阐述了生物正交化学在分子成像和药物传递等方面的研究与应用.     

Plasmodium berghei NK65 in the Inbred A/J Mouse: Immunity in the A/J Mouse Naturally Recovered from NK65C and Challenged with NK65E*

SYNOPSIS. Female retired breeder A/J mice were infected with Plasmodium berghei NK65C deme. Those animals which recovered were allowed to recrudesce and were inoculated again with NK65C. Twenty-one weeks after the original challenge, the mice were divided into 2 equal groups. One group was challenged with NK65C and the other with NK65E. Both demes of P. berghei were mosquito derived from NK65. NK65C appeared to give less protection to mice challenged with NK65E deme than to those challenged with the homologous NK65C deme. One mouse which had recovered from infection with NK65C deme died from the NK65E challenge. No definitive conclusions could be drawn regarding antigenic variation and virulence between demes E and C.