Plasmodium parasitophorous vacuole membrane-resident protein UIS4 manipulates host cell actin to avoid parasite elimination

© 2022 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Parasite-derived PVM-resident proteins are critical for complete parasite development inside hepatocytes, although the function of most of these proteins remains unknown. Here, we show that the upregulated in infectious sporozoites 4 (UIS4) protein, resident at the PVM, interacts with the host cell actin. By suppressing filamentous actin formation, UIS4 avoids parasite elimination. Host cell actin dynamics increases around UIS4-deficient parasites, which is associated with subsequent parasite elimination. Notably, parasite elimination is impaired significantly by the inhibition of host myosin-II, possibly through relieving the compression generated by actomyosin complexes at the host-parasite interface. Together, these data reveal that UIS4 has a critical role in the evasion of host defensive mechanisms, enabling hence EEF survival and development.This work was supported by grants from the Fundação para a Ciência e a Tecnologia (DRIVER-LISBOA-01-0145-FEDER-030751) and “la Caixa” Foundation (under the agreement HR17/52150010) to M.M.M. and Calouste Gulbenkian Foundation short project to V.M’B. and M.M.M.. V.M’B., A.L., and S.M. were sponsored by Fundação para a Ciência e a Tecnologia (SFRH/BD/113750/2015, PD/BD/114036/2015 and DL57/2016/CP1451/CT0010 respectively).info:eu-repo/semantics/publishedVersio

Expression in Yeast Links Field Polymorphisms in PfATP6 to in Vitro Artemisinin Resistance and Identifies New Inhibitor Classes

Background. The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. Results. Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. Conclusions. The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcom

Plasmodial sugar transporters as anti-malarial drug targets and comparisons with other protozoa

Glucose is the primary source of energy and a key substrate for most cells. Inhibition of cellular glucose uptake (the first step in its utilization) has, therefore, received attention as a potential therapeutic strategy to treat various unrelated diseases including malaria and cancers. For malaria, blood forms of parasites rely almost entirely on glycolysis for energy production and, without energy stores, they are dependent on the constant uptake of glucose. Plasmodium falciparum is the most dangerous human malarial parasite and its hexose transporter has been identified as being the major glucose transporter. In this review, recent progress regarding the validation and development of the P. falciparum hexose transporter as a drug target is described, highlighting the importance of robust target validation through both chemical and genetic methods. Therapeutic targeting potential of hexose transporters of other protozoan pathogens is also reviewed and discussed

Nutrient sensing modulates malaria parasite virulence

The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPKα, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence

Expression in Yeast Links Field Polymorphisms in PfATP6 to in Vitro Artemisinin Resistance and Identifies New Inhibitor Classes

Background. The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. Results. Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. Conclusions. The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcome

Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues

Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (−)-Epicatechin-gallate (ECG) and (−)-epigallocatechin-gallate (EGCG) inhibited d-glucose transport by GLUT1 and PfHT, and d-fructose transport by GLUT5, with apparent Ki values between 45 and 117 μM. BboHT1 was more potently inhibited by the ungallated catechins (−)-epicatechin (EC) and (−)-epigallocatechin (EGC), with apparent Ki values of 108 and 168 μM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external d-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents

Use of a Selective Inhibitor To Define the Chemotherapeutic Potential of the Plasmodial Hexose Transporter in Different Stages of the Parasite's Life Cycle▿

During blood infection, malarial parasites use d-glucose as their main energy source. The Plasmodium falciparum hexose transporter (PfHT), which mediates the uptake of d-glucose into parasites, is essential for survival of asexual blood-stage parasites. Recently, genetic studies in the rodent malaria model, Plasmodium berghei, found that the orthologous hexose transporter (PbHT) is expressed throughout the parasite's development within the mosquito vector, in addition to being essential during intraerythrocytic development. Here, using a d-glucose-derived specific inhibitor of plasmodial hexose transporters, compound 3361, we have investigated the importance of d-glucose uptake during liver and transmission stages of P. berghei. Initially, we confirmed the expression of PbHT during liver stage development, using a green fluorescent protein (GFP) tagging strategy. Compound 3361 inhibited liver-stage parasite development, with a 50% inhibitory concentration (IC50) of 11 μM. This process was insensitive to the external d-glucose concentration. In addition, compound 3361 inhibited ookinete development and microgametogenesis, with IC50s in the region of 250 μM (the latter in a d-glucose-sensitive manner). Consistent with our findings for the effect of compound 3361 on vector parasite stages, 1 mM compound 3361 demonstrated transmission blocking activity. These data indicate that novel chemotherapeutic interventions that target PfHT may be active against liver and, to a lesser extent, transmission stages, in addition to blood stages

Ca²⁺ tolerance of yeast mediated by PfCAX.

<p>(A) RT-PCR analysis of <i>pfcax</i> and <i>spfcax</i> expression in yeast compared with yeast transformed with the empty vector control. (B) Saturated liquid cultures of K665 (<i>pmc1 vcx1</i>) yeast transformed with <i>pfcax</i> in piHGpd, N-terminally truncated s<i>pfcax</i> in piHGpd, <i>scrcax1</i> in piHGpd and empty vector alone were serially diluted to the cell densities as indicated, then spotted onto selection medium lacking histidine (SD –His) and YPD medium containing 50 mM CaCl₂. Yeast growth at 30°C is shown after 4 days. A representative experiment is shown. (C) K665 yeast transformed with the various plasmids described in (B) were diluted to a cell density of 0.5 <i>A</i>₆₀₀ nm and inoculated into YPD medium containing concentrations of CaCl₂ from 25 to 150 mM. Yeast cell density was determined by absorbance measurements at 600 nm following growth shaking at 30°C for 16 h. Bars represent the mean ± SEM of 4 independent experiments (each with 8–12 replicates). Vector only, open bars; sCrCAX, closed bars; PfCAX, horizontally lined bars; sPfCAX, diagonally lined bars.</p