Rita Mukhopadhyay

Arsenite transport by mammalian aquaglyceroporins AQP7 and AQP9

Much is known about the transport of arsenite and antimonite into microbes, but the identities of mammalian transport proteins are unknown. The Saccharomyces cerevisiae FPS1 gene encodes a membrane protein homologous to the bacterial aquaglyceroporin GlpF and to mammalian aquaglyceroporins AQP7 and AQP9. Fps1p mediates glycerol uptake and glycerol efflux in response to hypoosmotic shock. Fps1p has been shown to facilitate uptake of the metalloids arsenite and antimonite, and the Escherichia coli homolog, GlpF, facilitates the uptake and sensitivity to metalloid salts. In this study, the ability of mammalian aquaglyceroporins AQP7 and AQP9 to substitute for the yeast Fps1p was examined. The fps1Δ strain of S. cerevisiae exhibits increased tolerance to arsenite and antimonite compared to a wild-type strain. Introduction of a plasmid containing AQP9 reverses the metalloid tolerance of the deletion strain. AQP7 was not expressed in yeast. The fps1Δ cells exhibit reduced transport of 73As(III) or 125Sb(III), but uptake is enhanced by expression of AQP9. Xenopus laevis oocytes microinjected with either AQP7 or AQP9 cRNA exhibited increased transport of 73As(III). These results suggest that AQP9 and AQP7 may be a major routes of arsenite uptake into mammalian cells, an observation potentially of large importance for understanding the action of arsenite as a human toxin and carcinogen, as well as its efficacy as a chemotherapeutic agent for acute promyelocytic leukemia.

Co-amplification of the gamma-glutamylcysteine synthetase gene gsh1 and of the ABC transporter gene pgpA in arsenite-resistant Leishmania tarentolae.

Resistance to the oxyanion arsenite in the parasite Leishmania is multifactorial. We have described previously the frequent amplification of the ABC transporter gene pgpA, the presence of a non‐PgpA thiol–metal efflux pump and increased levels of glutathione and trypanothione in resistant cells. Other loci are also amplified, although their role in resistance is unknown. By gene transfection, we have characterized one of these novel genes. It corresponds to gsh1, which encodes γ‐glutamylcysteine synthetase, an enzyme involved in the rate‐limiting step of glutathione biosynthesis. Transfection of gsh1 in wild‐type cells increased the levels of glutathione and trypanothione to levels found in resistant mutants. These transfectants were not resistant to metals. However, when gsh1 was transfected in partial revertants, it conferred resistance. As pgpA is frequently co‐amplified with gsh1, we co‐transfected the two genes into both wild‐type and partial revertants. Arsenite resistance levels in wild‐type cells could be accounted for by the contribution of PgpA alone. In the partial revertant, the gsh1 and pgpA gene product acted synergistically. These results support our previous suggestion that PgpA recognizes metals conjugated to thiols. Furthermore, amplification of gsh1 overcomes the rate‐limiting step in the synthesis of trypanothione, contributing to resistance. In addition, the results suggest that at least one more factor acts synergistically with the gsh1 gene product.

Leishmania major LmACR2 is a pentavalent antimony reductase that confers sensitivity to the drug pentostam.

Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases. To create the active form of the drug, Sb(V) must be reduced to Sb(III). Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase. The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major. LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae. Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes. LmACR2 was purified and shown to reduce both As(V) and Sb(V). This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania. We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.

Elevated levels of polyamines and trypanothione resulting from overexpression of the ornithine decarboxylase gene in arsenite-resistant Leishmania

The levels of trypanothione, a glutathione–spermidine conjugate, are increased in the protozoan parasite Leishmania selected for resistance to the heavy metal arsenite. The levels of putrescine and spermidine were increased in resistant mutants. This increase is mediated by overexpression of ornithine decarboxylase (ODC), the rate‐limiting enzyme in polyamine biosynthesis. Gene overexpression is generally mediated by gene amplification in Leishmania but, here, the mRNA and the enzymatic activity of ODC are increased without gene amplification. This RNA overexpression is stable when cells are grown in the absence of the drug and does not result from gene rearrangements or from an increased rate of RNA synthesis. Transient transfections suggest that mutations in the revertant cells contribute to these elevated levels of RNA. Stable transfection of the ODC gene increases the level of trypanothione, which can contribute to arsenite resistance. In addition to ODC overexpression, the gene for the ABC transporter PGPA is amplified in the mutants. The co‐transfection of the ODC and PGPA genes confers resistance in a synergistic fashion in partial revertants, also suggesting that PGPA recognizes metals conjugated to trypanothione.

Modulation in aquaglyceroporin AQP1 gene transcript levels in drug‐resistant Leishmania

Antimonial‐containing drugs are the first line of treatment against the parasite Leishmania. Resistance to antimonials has been correlated to its reduced accumulation. We used a dominant negative functional cloning strategy where a Leishmania mexicana expression cosmid bank was transfected in cells resistant to trivalent antimony (SbIII). Cells were selected for increased sensitivity to SbIII. One cosmid was isolated that could bestow SbIII sensitivity to resistant cells. The gene part of this cosmid that is responsible for increased SbIII sensitivity corresponds to AQP1, an aquaglyceroporin. AQP1 was recently shown to be a route by which SbIII can accumulate in Leishmania cells. Transport studies have shown that the L. mexicana AQP1 can restore SbIII transport in resistant cells. Southern blot analysis indicated that the copy number of neither the AQP1 gene nor the other AQP homologues was changed in antimony‐resistant mutants of several Leishmania species. The AQP1 gene sequence was also unchanged in mutants. However, the AQP1 RNA levels were downregulated in several Leishmania promastigote species resistant to antimonials. In general, but not always, the level of AQP1 transcript levels correlated well with the accumulation of SbIII and resistance levels in Leishmania cells. AQP1 thus appears to be a key determinant of antimonials accumulation and susceptibility in Leishmania.

Biochemical characterization of Leishmania major aquaglyceroporin LmAQP1: possible role in volume regulation and osmotaxis

The Leishmania major aquaglyceroporin, LmAQP1, is responsible for the transport of trivalent metalloids, arsenite and antimonite. We have earlier shown that downregulation of LmAQP1 provides resistance to trivalent antimony compounds whereas increased expression of LmAQP1 in drug‐resistant parasites can reverse the resistance. In this paper we describe the biochemical characterization of LmAQP1. Expression of LmAQP1 in Xenopus oocytes rendered them permeable to water, glycerol, methylglyoxal, dihydroxyacetone and sugar alcohols. The transport property of LmAQP1 was severely affected when a critical Arg230, located inside the pore of the channel, was altered to either alanine or lysine. Immunofluorescence and immuno‐electron microscopy revealed LmAQP1 to be localized to the flagellum of Leishmania promastigotes and in the flagellar pocket membrane and contractile vacuole/spongiome complex of amastigotes. This is the first report of an aquaglyceroporin being localized to the flagellum of any microbe. Leishmania promastigotes and amastigotes expressing LmAQP1 could regulate their volume in response to hypoosmotic stress. Additionally, Leishmania promastigotes overexpressing LmAQP1 were found to migrate faster towards an osmotic gradient. These results taken together suggest that Leishmania LmAQP1 has multiple physiological roles, being involved in solute transport, volume regulation and osmotaxis.

Reduced arsenic clearance and increased toxicity in aquaglyceroporin-9-null mice

Expressed in liver, aquaglyceroporin-9 (AQP9) is permeated by glycerol, arsenite, and other small, neutral solutes. To evaluate a possible protective role, AQP9-null mice were evaluated for in vivo arsenic toxicity. After injection with NaAsO2, AQP9-null mice suffer reduced survival rates (LD50, 12 mg/kg) compared with WT mice (LD50, 15 mg/kg). The highest tissue level of arsenic is in heart, with AQP9-null mice accumulating 10–20 times more arsenic than WT mice. Within hours after NaAsO2 injection, AQP9-null mice sustain profound bradycardia, despite normal serum electrolytes. Increased arsenic levels are also present in liver, lung, spleen, and testis of AQP9-null mice. Arsenic levels in the feces and urine of AQP9-null mice are only ≈10% of the WT levels, and reduced clearance of multiple arsenic species by the AQP9-null mice suggests that AQP9 is involved in the export of multiple forms of arsenic. Immunohistochemical staining of liver sections revealed that AQP9 is most abundant in basolateral membrane of hepatocytes adjacent to the sinusoids. AQP9 is not detected in heart or kidney by PCR or immunohistochemistry. We propose that AQP9 provides a route for excretion of arsenic by the liver, thereby providing partial protection of the whole animal from arsenic toxicity.

Aquaglyceroporins: ancient channels for metalloids

The identification of aquaglyceroporins as uptake channels for arsenic and antimony shows how these toxic elements can enter the food chain, and suggests that food plants could be genetically modified to exclude arsenic while still accumulating boron and silicon.

Aquaglyceroporins and metalloid transport: Implications in human diseases

Aquaglyceroporin (AQP) channels facilitate the diffusion of a wide range of neutral solutes, including water, glycerol, and other small uncharged solutes. More recently, AQPs have been shown to allow the passage of trivalent arsenic and antimony compounds. Arsenic and antimony are metalloid elements. At physiological pH, the trivalent metalloids behave as molecular mimics of glycerol, and are conducted through AQP channels. Arsenicals and antimonials are extremely toxic to cells. Despite their toxicity, both metalloids are used as chemotherapeutic agents for the treatment of cancer and protozoan parasitic diseases. The metalloid homeostasis property of AQPs can be a mixed blessing. In some cases, AQPs form part of the detoxification pathway, and extrude metalloids from cells. In other instances, AQPs allow the transport of metalloids into cells, thereby conferring sensitivity. Understanding the factors that modulate AQP expression will aid in a better understanding of metalloid toxicity and also provide newer approaches to metalloid based chemotherapy.

Targeted disruption of the mouse Asna1 gene results in embryonic lethality.

The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Homologues of the bacterial ArsA ATPase are widespread in nature. We had earlier identified the mouse homologue (Asna1) that exhibits 27% identity to the bacterial ArsA ATPase. To identify the physiological role of the protein, heterozygous Asna1 knockout mice (Asna1 +/−) were generated by homologous recombination. The Asna1 +/− mice displayed similar phenotype as the wild‐type mice. However, early embryonic lethality was observed in homozygous Asna1 knockout embryos, between E3.5 (E = embryonic day) and E8.5 stage. These findings indicate that Asna1 plays a crucial role during early embryonic development.