Structure and non-essential function of glycerol kinase in <i>Plasmodium</i> <i>falciparum</i> blood stages

Malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). Microarray analysis identified glycerol kinase (GK) as the second most highly upregulated gene in Plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. Phosphorylation of glycerol by GK is the rate-limiting step in glycerol utilization. Deletion of this gene from P. falciparum had no effect on asexual parasite growth, but surprisingly also had no effect on gametocyte development or exflagellation, suggesting that these life cycle stages do not utilize host-derived glycerol as a carbon source. Kinetic studies of purified PfGK showed that the enzyme is not regulated by fructose 1,6 bisphosphate. The high-resolution crystal structure of P. falciparum GK, the first of a eukaryotic GK, reveals two domains embracing a capacious ligand-binding groove. In the complexes of PfGK with glycerol and ADP, we observed closed and open forms of the active site respectively. The 27° domain opening is larger than in orthologous systems and exposes an extensive surface with potential for exploitation in selective inhibitor design should the enzyme prove to be essential in vivo either in the human or in the mosquito

Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity

Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for melarsoprol and pentamidine-uptake, and melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to melarsoprol and respiratory inhibitors described here might be exploited

Aquaglyceroporin PbAQP during intraerythrocytic development of the malaria parasite Plasmodium berghei

The malaria parasite can use host plasma glycerol for lipid biosynthesis and membrane biogenesis during the asexual intraerythrocytic development. The molecular basis for glycerol uptake into the parasite is undefined. We hypothesize that the Plasmodium aquaglyceroporin provides the pathway for glycerol uptake into the malaria parasite. To test this hypothesis, we identified the orthologue of Plasmodium falciparum aquaglyceroporin (PfAQP) in the rodent malaria parasite, Plasmodium berghei (PbAQP), and examined the biological role of PbAQP by performing a targeted deletion of the PbAQP gene. PbAQP and PfAQP are 62% identical in sequence. In contrast to the canonical NPA (Asn-Pro-Ala) motifs in most aquaporins, the PbAQP has NLA (Asn-Leu-Ala) and NPS (Asn-Leu-Ser) in those positions. PbAQP expressed in Xenopus oocytes was permeable to water and glycerol, suggesting that PbAQP is an aquaglyceroporin. In P. berghei, PbAQP was localized to the parasite plasma membrane. The PbAQP-null parasites were viable; however, they were highly deficient in glycerol transport. In addition, they proliferated more slowly compared with the WT parasites, and mice infected with PbAQP-null parasites survived longer. Taken together, these findings suggest that PbAQP provides the pathway for the entry of glycerol into P. berghei and contributes to the growth of the parasite during the asexual intraerythrocytic stages of infection. In conclusion, we demonstrate here that PbAQP plays an important role in the blood-stage development of the rodent malaria parasite during infection in mice and could be added to the list of targets for the design of antimalarial drugs

Aquaporin 9 is the major pathway for glycerol uptake by mouse erythrocytes, with implications for malarial virulence

Human and rodent erythrocytes are known to be highly permeable to glycerol. Aquaglyceroporin aquaporin (AQP)3 is the major glycerol channel in human and rat erythrocytes. However, AQP3 expression has not been observed in mouse erythrocytes. Here we report the presence of an aquaglyceroporin, AQP9, in mouse erythrocytes. AQP9 levels rise as reticulocytes mature into erythrocytes and as neonatal pups develop into adult mice. Mice bearing targeted disruption of both alleles encoding AQP9 have erythrocytes that appear morphologically normal. Compared with WT cells, erythrocytes from AQP9-null mice are defective in rapid glycerol transport across the cell membrane when measured by osmotic lysis, [(14)C]glycerol uptake, or stopped-flow light scattering. In contrast, the water and urea permeabilities are intact. Although the physiological role of glycerol in the normal function of erythrocytes is not clear, plasma glycerol is an important substrate for lipid biosynthesis of intraerythrocytic malarial parasites. AQP9-null mice at the age of 4 months infected with Plasmodium berghei survive longer during the initial phase of infection compared with WT mice. We conclude that AQP9 is the major glycerol channel in mouse erythrocytes and suggest that this transport pathway may contribute to the virulence of intraerythrocytic stages of malarial infection

Immunohistochemical localization and mRNA expression of aquaporins in the macula utriculi of patients with Meniere’s disease and acoustic neuroma

Meniere’s disease is nearly invariably associated with endolymphatic hydrops (the net accumulation of water in the inner ear endolymphatic space). Vestibular maculae utriculi were acquired from patients undergoing surgery for Meniere’s disease and acoustic neuroma and from autopsy (subjects with normal hearing and balance). Quantitative immunostaining was conducted with antibodies against aquaporins (AQPs) 1, 4, and 6, Na+K+ATPase, Na+K+2Cl co-transporter (NKCC1), and α-syntrophin. mRNA was extracted from the surgically acquired utricles from subjects with Meniere’s disease and acoustic neuroma to conduct quantitative real-time reverse transcription with polymerase chain reaction for AQP1, AQP4, and AQP6. AQP1 immunoreactivity (−IR) was located in blood vessels and fibrocytes in the underlying stroma, without any apparent alteration in Meniere’s specimens when compared with acoustic neuroma and autopsy specimens. AQP4-IR localized to the epithelial basolateral supporting cells in Meniere’s disease, acoustic neuroma, and autopsy. In specimens from subjects with Meniere’s disease, AQP4-IR was significantly decreased compared with autopsy and acoustic neuroma specimens. AQP6-IR occurred in the sub-apical vestibular supporting cells in acoustic neuroma and autopsy samples. However, in Meniere’s disease specimens, AQP6-IR was significantly increased and diffusely redistributed throughout the supporting cell cytoplasm. Na+K+ATPase, NKCC1, and α-syntrophin were expressed within sensory epithelia and were unaltered in Meniere’s disease specimens. Expression of AQP1, AQP4, or AQP6 mRNA did not differ in vestibular endorgans from patients with Meniere’s disease. Changes in AQP4 (decreased) and AQP6 (increased) expression in Meniere’s disease specimens suggest that the supporting cell might be a cellular target

Induction of metabolic acidosis with ammonium chloride (NH4Cl) in mice and rats-species differences and technical considerations

Ammonium chloride addition to drinking water is often used to induce metabolic acidosis (MA) in rodents but may also cause mild dehydration. Previous microarray screening of acidotic mouse kidneys showed upregulation of genes involved in renal water handling. Thus, we compared two protocols to induce metabolic acidosis in mice and rats: standard 0.28M NH(4)Cl in drinking water or an equivalent amount of NH(4)Cl in food. Both treatments induced MA in mice and rats. In rats, NH (4)Cl in water caused signs of dehydration, reduced mRNA abundance of the vasopression receptor 2 (V2R), increased protein abundance of the aquaporin water channels AQP2 and AQP3 and stimulated phosphorylation of AQP2 at residues Ser256 and Ser261. In contrast, NH(4)Cl in food induced massive diuresis, decreased mRNA levels of V2R, AQP2, and AQP3, did not affect protein abundance of AQP2 and AQP3, and stimulated phosphorylation at Ser261 but not pSer256 of AQP2. In mice, NH(4)Cl in drinking water stimulated urine concentration, increased AQP2 and V2R mRNA levels, and enhanced AQP2 and AQP3 protein expression with higher levels of AQP2 pSer256 and pSer261. Addition of NH(4)Cl to food, stimulated diuresis, had no effect on mRNA levels of AQP2, AQP3, and V2R, and enhanced only AQP3 protein abundance whereas pSer256-AQP2 and pSer261-AQP2 remained unaltered. Similarly, AQP2 staining was more intense and luminal in kidney from mice with NH(4)Cl in water but not in food. Pendrin, SNAT3 and PEPCK mRNA expression in mouse kidney were not affected by the route of N(4)Cl application. Thus, addition of NH(4)Cl to water or food causes MA but has differential effects on diuresis and expression of mRNAs and proteins related to renal water handling. Moreover, mice and rats respond differently to NH(4)Cl loading, and increased water intake and diuresis may be a compensatory mechanism during MA. It may be necessary to consider these effects in planning and interpreting experiments of NH(4)Cl supplementation to animals

Aquaglyceroporin PbAQP is required for efficient progression through the liver stage of Plasmodium infection

Abstract The discovery of aquaglyceroporins (AQP) has highlighted a new mechanism of membrane solute transport that may hold therapeutic potential for controlling parasitic infections, including malaria. Plasmodium parasites express a single AQP at the plasma membrane that functions as a channel for water, nutrients and waste into and out cells. We previously demonstrated that Plasmodium berghei targeted for PbAQP deletion are deficient in glycerol import and less virulent than wild-type parasites during the blood developmental stage. Here, we have examined the contribution of PbAQP to the infectivity of P. berghei in the liver. PbAQP is expressed in the sporozoite mosquito stage and is detected at low levels in intrahepatic parasites at the onset of hepatocyte infection. As the parasites progress to late hepatic stages, PbAQP transcription increases and PbAQP localizes to the plasma membrane of hepatic merozoites. Compared to wild-type parasites, PbAQP-null sporozoites exhibit a delay in blood stage infection due to slower replication in hepatocytes, resulting in retardation of merosome production. Furthermore, PbAQP disruption results in a significant reduction in erythrocyte infectivity by hepatocyte-derived merozoites. Hepatic merozoites incorporate exogenous glycerol into glycerophospholipids and PbAQP-null merozoites contain less phosphatidylcholine than wild-type merozoites, underlining the contribution of Plasmodium AQP to phospholipid syntheses