Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum

Abstract Background The development of differentiated sexual stages (gametocytes) within human red blood cells is essential for the propagation of the malaria parasite, since only mature gametocytes will survive in the mosquito’s midgut. Hence gametocytogenesis is a pre-requisite for transmission of the disease. Physiological changes involved in sexual differentiation are still enigmatic. In particular the lipid metabolism—despite being central to cellular regulation and development—is not well explored. Methods Here the lipid profiles of red blood cells infected with the five different sexual stages of Plasmodium falciparum were analysed by mass spectrometry and compared to those from uninfected and asexual trophozoite infected erythrocytes. Results Fundamental differences between erythrocytes infected with the different parasite stages were revealed. In mature gametocytes many lipids that decrease in the trophozoite and early gametocyte infected red blood cells are regained. In particular, regulators of membrane fluidity, cholesterol and sphingomyelin, increased significantly during gametocyte maturation. Neutral lipids (serving mainly as caloriometric reserves) increased from 3 % of total lipids in uninfected to 27 % in stage V gametocyte infected red blood cells. The major membrane lipid class (phospholipids) decreased during gametocyte development. Conclusions The lipid profiles of infected erythrocytes are characteristic for the particular parasite life cycle and maturity stages of gametocytes. The obtained lipid profiles are crucial in revealing the lipid metabolism of malaria parasites and identifying targets to interfere with this deadly disease.We are grateful to the Australian Red Cross for providing human RBCs and serum. Support of the Australian Research Council is acknowledged. TWM is an Australian Research Council Future Fellow (FT110100249)

A G358S mutation in the Plasmodium falciparum Na⁺ pump PfATP4 confers clinically-relevant resistance to cipargamin

Diverse compounds target the Plasmodium falciparum Na(+) pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4(G358S) parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na(+) regulation. The G358S mutation reduces the affinity of PfATP4 for Na(+) and is associated with an increase in the parasite’s resting cytosolic [Na(+)]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4(G358S) parasites, and that their combination with unrelated antimalarials may mitigate against resistance development

Human dihydrofolate reductase influences the sensitivity of the malaria parasite Plasmodium falciparum to ketotifen – A cautionary tale in screening transgenic parasites

Ketotifen has recently been reported to inhibit the growth of both asexual and sexual malaria parasites. A parasite transporter, PfgABCG2, has been implicated in its mechanism of action. Human dihydrofolate reductase (hDHFR) is the most commonly used selectable marker to create transgenic Plasmodium falciparum cell lines. Growth assays using transgenic P. falciparum parasites with different selectable markers revealed that the presence of hDHFR rather than the absence of PfgABCG2 is responsible for a shift in the parasite's sensitivity to ketotifen. Employing a range of in vitro assays and liquid chromatography-mass spectrometry we show that ketotifen influences hDHFR activity, but it is not metabolised by the enzyme. Our data also highlights potential pitfalls when functionally characterising transgenic parasites

On the pathway to better birth outcomes? A systematic review of azithromycin and curable sexually transmitted infections.

The WHO recommends the administration of sulfadoxine-pyrimethamine (SP) to all pregnant women living in areas of moderate (stable) to high malaria transmission during scheduled antenatal visits, beginning in the second trimester and continuing to delivery. Malaria parasites have lost sensitivity to SP in many endemic areas, prompting the investigation of alternatives that include azithromycin-based combination (ABC) therapies. Use of ABC therapies may also confer protection against curable sexually transmitted infections and reproductive tract infections (STIs/RTIs). The magnitude of protection at the population level would depend on the efficacy of the azithromycin-based regimen used and the underlying prevalence of curable STIs/RTIs among pregnant women who receive preventive treatment. This systematic review summarizes the efficacy data of azithromycin against curable STIs/RTIs

A novel epileptic encephalopathy mutation in KCNB1 disrupts Kv2.1 ion selectivity, expression, and localization

The epileptic encephalopathies are a group of highly heterogeneous genetic disorders. The majority of disease-causing mutations alter genes encoding voltage-gated ion channels, neurotransmitter receptors, or synaptic proteins. We have identified a novel de novo pathogenic K(+) channel variant in an idiopathic epileptic encephalopathy family. Here, we report the effects of this mutation on channel function and heterologous expression in cell lines. We present a case report of infantile epileptic encephalopathy in a young girl, and trio-exome sequencing to determine the genetic etiology of her disorder. The patient was heterozygous for a de novo missense variant in the coding region of the KCNB1 gene, c.1133T>C. The variant encodes a V378A mutation in the α subunit of the Kv2.1 voltage-gated K(+) channel, which is expressed at high levels in central neurons and is an important regulator of neuronal excitability. We found that expression of the V378A variant results in voltage-activated currents that are sensitive to the selective Kv2 channel blocker guangxitoxin-1E. These voltage-activated Kv2.1 V378A currents were nonselective among monovalent cations. Striking cell background–dependent differences in expression and subcellular localization of the V378A mutation were observed in heterologous cells. Further, coexpression of V378A subunits and wild-type Kv2.1 subunits reciprocally affects their respective trafficking characteristics. A recent study reported epileptic encephalopathy-linked missense variants that render Kv2.1 a tonically activated, nonselective cation channel that is not voltage activated. Our findings strengthen the correlation between mutations that result in loss of Kv2.1 ion selectivity and development of epileptic encephalopathy. However, the strong voltage sensitivity of currents from the V378A mutant indicates that the loss of voltage-sensitive gating seen in all other reported disease mutants is not required for an epileptic encephalopathy phenotype. In addition to electrophysiological differences, we suggest that defects in expression and subcellular localization of Kv2.1 V378A channels could contribute to the pathophysiology of this KCNB1 variant