RESUMEN
Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation-a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.
Asunto(s)
Antimaláricos , Antimaláricos/farmacología , Indoles/farmacología , Chaperonas Moleculares , Plasmodium falciparumRESUMEN
Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.
Asunto(s)
Antimaláricos/uso terapéutico , Descubrimiento de Drogas , Malaria/tratamiento farmacológico , Malaria/transmisión , Pandemias , Aedes/parasitología , Animales , Antimaláricos/química , Antimaláricos/farmacología , Análisis por Conglomerados , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Estadios del Ciclo de Vida/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/parasitología , Malaria/epidemiología , Masculino , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
The antibiotic actinonin kills malaria parasites (Plasmodium falciparum) by interfering with apicoplast function. Early evidence suggested that actinonin inhibited prokaryote-like post-translational modification in the apicoplast; mimicking its activity against bacteria. However, Amberg Johnson et al. (2017) identified the metalloprotease TgFtsH1 as the target of actinonin in the related parasite Toxoplasma gondii and implicated P. falciparum FtsH1 as a likely target in malaria parasites. The authors were not, however, able to recover actinonin resistant malaria parasites, leaving the specific target of actinonin uncertain. We generated actinonin resistant P. falciparum by in vitro selection and identified a specific sequence change in PfFtsH1 associated with resistance. Introduction of this point mutation using CRISPr-Cas9 allelic replacement was sufficient to confer actinonin resistance in P. falciparum. Our data unequivocally identify PfFtsH1 as the target of actinonin and suggests that actinonin should not be included in the highly valuable collection of 'irresistible' drugs for combatting malaria.
Asunto(s)
Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Proteínas de la Membrana/genética , Metaloproteasas/genética , Plasmodium falciparum/efectos de los fármacos , Mutación Puntual , Proteínas Protozoarias/genética , Ácidos Hidroxámicos/farmacología , Proteínas de la Membrana/metabolismo , Metaloproteasas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.
Asunto(s)
Plasmodium falciparum/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Artemisininas/farmacología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Moleculares , Simulación de Dinámica Molecular , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Complejo de la Endopetidasa Proteasomal/genética , Conformación Proteica , Multimerización de Proteína , Proteostasis , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Apicomplexan parasites possess a plastid organelle called the apicoplast. Inhibitors that selectively target apicoplast housekeeping functions, including DNA replication and protein translation, are lethal for the parasite, and several (doxycycline, clindamycin, and azithromycin) are in clinical use as antimalarials. A major limitation of such drugs is that treated parasites only arrest one intraerythrocytic development cycle (approximately 48 hours) after treatment commences, a phenotype known as the 'delayed death' effect. The molecular basis of delayed death is a long-standing mystery in parasitology, and establishing the mechanism would aid rational clinical implementation of apicoplast-targeted drugs. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells and cannot produce the sole, blood-stage essential metabolic product of the apicoplast: the isoprenoid precursor isopentenyl-pyrophosphate. How the isoprenoid precursor depletion kills the parasite remains unknown. We investigated the requirements for the range of isoprenoids in the human malaria parasite Plasmodium falciparum and characterised the molecular and morphological phenotype of parasites experiencing delayed death. Metabolomic profiling reveals disruption of digestive vacuole function in the absence of apicoplast derived isoprenoids. Three-dimensional electron microscopy reveals digestive vacuole fragmentation and the accumulation of cytostomal invaginations, characteristics common in digestive vacuole disruption. We show that digestive vacuole disruption results from a defect in the trafficking of vesicles to the digestive vacuole. The loss of prenylation of vesicular trafficking proteins abrogates their membrane attachment and function and prevents the parasite from feeding. Our data show that the proximate cause of delayed death is an interruption of protein prenylation and consequent cellular trafficking defects.
Asunto(s)
Apicoplastos/metabolismo , Espacio Intracelular/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Antimaláricos/farmacología , Muerte Celular/efectos de los fármacos , Hemiterpenos/metabolismo , Hemiterpenos/farmacología , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/parasitología , Malaria Falciparum/parasitología , Metabolómica/métodos , Compuestos Organofosforados/metabolismo , Compuestos Organofosforados/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/fisiología , Prenilación de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , Vacuolas/parasitologíaRESUMEN
The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome ß5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.
Asunto(s)
Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Diseño de Fármacos , Plasmodium falciparum/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Línea Celular , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos/efectos de los fármacos , Humanos , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/químicaRESUMEN
Artemisinin and its derivatives (collectively referred to as ARTs) rapidly reduce the parasite burden in Plasmodium falciparum infections, and antimalarial control is highly dependent on ART combination therapies (ACTs). Decreased sensitivity to ARTs is emerging, making it critically important to understand the mechanism of action of ARTs. Here we demonstrate that dihydroartemisinin (DHA), the clinically relevant ART, kills parasites via a two-pronged mechanism, causing protein damage, and compromising parasite proteasome function. The consequent accumulation of proteasome substrates, i.e., unfolded/damaged and polyubiquitinated proteins, activates the ER stress response and underpins DHA-mediated killing. Specific inhibitors of the proteasome cause a similar build-up of polyubiquitinated proteins, leading to parasite killing. Blocking protein synthesis with a translation inhibitor or inhibiting the ubiquitin-activating enzyme, E1, reduces the level of damaged, polyubiquitinated proteins, alleviates the stress response, and dramatically antagonizes DHA activity.
RESUMEN
The malaria parasite exports numerous proteins into its host red blood cell (RBC). The trafficking of these exported effectors is complex. Proteins are first routed through the secretory system, into the parasitophorous vacuole (PV), a membranous compartment enclosing the parasite. Proteins are then translocated across the PV membrane in a process requiring ATP and unfolding. Once in the RBC compartment the exported proteins are then refolded and further trafficked to their final localizations. Chaperones are important in the unfolding and refolding processes. Recently, it was suggested that the parasite TRiC chaperonin complex is exported, and that it is involved in trafficking of exported effectors. Using a parasite-specific antibody and epitope-tagged transgenic parasites we could observe no export of Plasmodium TRiC into the RBC. We tested the importance of the parasite TRiC by creating a regulatable knockdown line of the TRiC-θ subunit. Loss of the parasite TRiC-θ led to a severe growth defect in asexual development, but did not alter protein export into the RBC. These observations indicate that the TRiC proteins play a critical role in parasite biology, though their function, within the parasite, appears unrelated to protein trafficking in the RBC compartment.
Asunto(s)
Chaperoninas/metabolismo , Citosol/metabolismo , Malaria Falciparum/patología , Complejos Multiproteicos/metabolismo , Plasmodium falciparum/patogenicidad , Membrana Celular/metabolismo , Eritrocitos/parasitología , Regulación de la Expresión Génica/genética , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Replegamiento Proteico , Transporte de Proteínas/fisiología , Vacuolas/parasitologíaRESUMEN
Erythrocytes are reservoirs of important epoxide-containing lipid signaling molecules, including epoxyeicosatrienoic acids (EETs). EETs function as vasodilators and anti-inflammatory modulators in the bloodstream. Bioactive EETs are hydrolyzed to less active diols (dihydroxyeicosatrienoic acids) by epoxide hydrolases (EHs). The malaria parasite Plasmodium falciparum infects host red blood cells (RBCs) and exports hundreds of proteins into the RBC compartment. In this study, we show that two parasite epoxide hydrolases, P falciparum epoxide hydrolases 1 (PfEH1) and 2 (PfEH2), both with noncanonical serine nucleophiles, are exported to the periphery of infected RBCs. PfEH1 and PfEH2 were successfully expressed in Escherichia coli, and they hydrolyzed physiologically relevant erythrocyte EETs. Mutations in active site residues of PfEH1 ablated the ability of the enzyme to hydrolyze an epoxide substrate. Overexpression of PfEH1 or PfEH2 in parasite-infected RBCs resulted in a significant alteration in the epoxide fatty acids stored in RBC phospholipids. We hypothesize that the parasite disruption of epoxide-containing signaling lipids leads to perturbed vascular function, creating favorable conditions for binding and sequestration of infected RBCs to the microvascular endothelium. IMPORTANCE: The malaria parasite exports hundreds of proteins into the erythrocyte compartment. However, for most of these proteins, their physiological function is unknown. In this study, we investigate two "hypothetical" proteins of the α/ß-hydrolase fold family that share sequence similarity with epoxide hydrolases (EHs)-enzymes that destroy bioactive epoxides. Altering EH expression in parasite-infected erythrocytes resulted in a significant change in the epoxide fatty acids stored in the host cell. We propose that these EH enzymes may help the parasite to manipulate host blood vessel opening and inflame the vessel walls as they pass through the circulation system. Understanding how the malaria parasite interacts with its host RBCs will aid in our ability to combat this deadly disease.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Epóxido Hidrolasas/metabolismo , Eritrocitos/química , Eritrocitos/parasitología , Metabolismo de los Lípidos , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Sustitución de Aminoácidos , Dominio Catalítico , Clonación Molecular , Análisis Mutacional de ADN , Epóxido Hidrolasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Malaria Falciparum/patología , Plasmodium falciparum/genética , Proteínas Protozoarias/genéticaRESUMEN
The intraerythrocytic malaria parasite, Plasmodium falciparum, maintains a low cytosolic Na(+) concentration and the plasma membrane P-type cation translocating ATPase 'PfATP4' has been implicated as playing a key role in this process. PfATP4 has been the subject of significant attention in recent years as mutations in this protein confer resistance to a growing number of new antimalarial compounds, including the spiroindolones, the pyrazoles, the dihydroisoquinolones, and a number of the antimalarial agents in the Medicines for Malaria Venture's 'Malaria Box'. On exposure of parasites to these compounds there is a rapid disruption of cytosolic Na(+). Whether, and if so how, such chemically distinct compounds interact with PfATP4, and how such interactions lead to parasite death, is not yet clear. The fact that multiple different chemical classes have converged upon PfATP4 highlights its significance as a potential target for new generation antimalarial agents. A spiroindolone (KAE609, now known as cipargamin) has progressed through Phase I and IIa clinical trials with favourable results. In this review we consider the physiological role of PfATP4, summarise the current repertoire of antimalarial compounds for which PfATP4 is implicated in their mechanism of action, and provide an outlook on translation from target identification in the laboratory to patient treatment in the field.
Asunto(s)
Antimaláricos/farmacología , ATPasas Transportadoras de Calcio/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Malaria Falciparum/parasitología , Plasmodium falciparum/efectos de los fármacos , ATPasas Transportadoras de Calcio/genética , Eritrocitos/metabolismo , Eritrocitos/parasitología , Humanos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/enzimologíaRESUMEN
Phylum Apicomplexa comprises a large group of obligate intracellular parasites of high medical and veterinary importance. These organisms succeed intracellularly by effecting remarkable changes in a broad range of diverse host cells. The transformation of the host erythrocyte is particularly striking in the case of the malaria parasite Plasmodium falciparum. P. falciparum exports hundreds of proteins that mediate a complex cellular renovation marked by changes in the permeability, rigidity, and cytoadherence properties of the host erythrocyte. The past decade has seen enormous progress in understanding the identity and function of these exported effectors, as well as the mechanisms by which they are trafficked into the host cell. Here we review these advances, place them in the context of host manipulation by related apicomplexans, and propose key directions for future research.
Asunto(s)
Eritrocitos/parasitología , Plasmodium/fisiología , Animales , Apicomplexa/clasificación , Apicomplexa/fisiología , Humanos , Malaria/inmunología , Malaria/parasitología , Señales de Clasificación de Proteína , Proteínas/metabolismo , Infecciones por Protozoos/inmunología , Infecciones por Protozoos/parasitología , Proteínas Protozoarias/metabolismoRESUMEN
Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na(+) levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na(+) homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.
Asunto(s)
Antimaláricos/farmacología , ATPasas Transportadoras de Calcio/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Isoquinolinas/farmacología , Malaria/tratamiento farmacológico , Modelos Moleculares , Plasmodium/efectos de los fármacos , Antimaláricos/farmacocinética , ATPasas Transportadoras de Calcio/genética , Senescencia Celular/efectos de los fármacos , Descubrimiento de Drogas , Resistencia a Medicamentos/genética , Eritrocitos/efectos de los fármacos , Citometría de Flujo , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Ensayos Analíticos de Alto Rendimiento , Isoquinolinas/farmacocinética , Estructura MolecularRESUMEN
The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na(+) homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.
Asunto(s)
Amidas/farmacología , Antimaláricos/farmacología , Bencimidazoles/farmacología , Eritrocitos/parasitología , Malaria/parasitología , Plasmodium falciparum/efectos de los fármacos , Pirazoles/farmacología , Sodio/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Femenino , Homeostasis/efectos de los fármacos , Humanos , Masculino , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas ProtozoariasRESUMEN
The intraerythrocytic malaria parasite has, on its plasma membrane, a H(+)-extruding V-type H(+)-ATPase that plays a central role in maintaining the resting cytosolic pH at around 7.3. Previous studies have demonstrated the presence in the parasite of an unknown acidification mechanism that is revealed on inhibition of the V-type H(+)-ATPase. Here we show that this acidification is dependent on the presence of extracellular Na(+), and is associated with the activity of a plasma membrane Na(+)-ATPase that is inhibited by the novel antimalarial spiroindolone NITD246 and is postulated to export Na(+) ions in counter-transport with H(+) ions. The proposed import of H(+) by the Na(+)-extruding Na(+)-ATPase necessitates "abundant H(+) pumping" by the V-type H(+)-ATPase (Ginsburg H. Abundant proton pumping in Plasmodium falciparum, but why? Trends in Parasitology 2002;18:483-6) and has significant implications for the energy budget of the parasite.
Asunto(s)
Ácidos/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Plasmodium falciparum/efectos de los fármacos , ATPasas de Translocación de Protón/metabolismo , Sodio/metabolismo , Antimaláricos/metabolismoRESUMEN
The malaria parasite Plasmodium falciparum establishes in the host erythrocyte plasma membrane new permeability pathways that mediate nutrient uptake into the infected cell. These pathways simultaneously allow Na(+) influx, causing [Na(+)] in the infected erythrocyte cytosol to increase to high levels. The intraerythrocytic parasite itself maintains a low cytosolic [Na(+)] via unknown mechanisms. Here we present evidence that the intraerythrocytic parasite actively extrudes Na(+) against an inward gradient via PfATP4, a parasite plasma membrane protein with sequence similarities to Na(+)-ATPases of lower eukaryotes. Mutations in PfATP4 confer resistance to a potent class of antimalarials, the spiroindolones. Consistent with this, the spiroindolones cause a profound disruption in parasite Na(+) homeostasis, which is attenuated in parasites bearing resistance-conferring mutations in PfATP4. The mutant parasites also show some impairment of Na(+) regulation. Taken together, our results are consistent with PfATP4 being a Na(+) efflux ATPase and a target of the spiroindolones.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Antimaláricos/farmacología , Proteínas de Transporte de Catión/metabolismo , Plasmodium falciparum/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Adenosina Trifosfatasas/genética , Proteínas de Transporte de Catión/genética , Resistencia a Medicamentos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Homeostasis , Humanos , Indoles/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Ouabaína/farmacología , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Trofozoítos/efectos de los fármacos , Trofozoítos/metabolismoRESUMEN
The intraerythrocytic malaria parasite, Plasmodium falciparum maintains an intracellular pH (pH(i)) of around 7.3. If subjected to an experimentally imposed acidification the parasite extrudes H(+), thereby undergoing a pH(i) recovery. In a recent study, Bennett et al. [Bennett TN, Patel J, Ferdig MT, Roepe PD. P. falciparum Na(+)/H(+) exchanger activity and quinine resistance. Mol Biochem Parasitol 2007;153:48-58] used the H(+) ionophore nigericin, in conjunction with an acidic medium, to acidify the parasite cytosol, and then used bovine serum albumin (BSA) to scavenge the nigericin from the parasite membrane. The ensuing Na(+)-dependent pH(i) recovery, seen following an increase in the extracellular pH, was attributed to a plasma membrane Na(+)/H(+) exchanger. This is at odds with previous reports that the primary H(+) extrusion mechanism in the parasite is a plasma membrane V-type H(+)-ATPase. Here we present evidence that the Na(+)-dependent efflux of H(+) from parasites acidified using nigericin/BSA is attributable to Na(+)/H(+) exchange via residual nigericin remaining in the parasite plasma membrane, rather than to endogenous transporter activity.