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

Species-Specific Antimonial Sensitivity in Leishmania Is Driven by Post-Transcriptional Regulation of AQP1

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3’-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species

UDP-galactose 4-epimerase from Kluyveromyces fragilis : Reconstitution of Holoenzyme Structure After Dissociation With Parachloromercuribenzoate

UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis (Kluyveromyces marxianus var. marxianus) is a homodimer of molecular mass 75 kDa/subunit and has one mol NAD firmly bound/dimer. The pathway for the assembly of the holoenzyme structure has been studied after dissociating the native epimerase with p-chloromercuribenzoate into inactive mercurated monomers. The process of dissociation was not associated with unfolding of the molecules. Reconstitution of the functional holoenzyme was done by reduction with dithiothreitol and addition of extra NAD. The reaction was thus followed to monitor maturation of the enzyme from the folded monomeric state. The reconstituted enzyme was similar to the native enzyme in terms of a number of physiochemical properties such as secondary, tertiary and quarternary structures, Km for the substrate UDP-galactose, reductive inhibition, interaction with the fluorophore 1-anilino 8-naphthalene sulphonic acid (ANS), etc. Reconstitution under low ionic strength buffer (I 5 0.011) shows that the presence of NAD is essential for the formation of a dimeric structure. However, dimeric apoenzyme could also be stabilized under high ionic strength buffer (I 5 0.1). Reactivation was strongly dependent on pH, being most effective at pH 8.1. Kinetic evidence suggested that, at low ionic strength, assembly of NAD over dimeric apoenzyme is the rate-limiting step in expressing catalytic activity. This process has a low energy of activation of 27.2 kJ/mol

Novel Pathway for Arsenic Detoxification in the Legume Symbiont Sinorhizobium meliloti

We report a novel pathway for arsenic detoxification in the legume symbiont Sinorhizobium meliloti. Although a majority of ars operons consist of three genes, arsR (transcriptional regulator), arsB [As(OH)(3)/H(+) antiporter], and arsC (arsenate reductase), the S. meliloti ars operon includes an aquaglyceroporin (aqpS) in place of arsB. The presence of AqpS in an arsenic resistance operon is interesting, since aquaglyceroporin channels have previously been shown to adventitiously facilitate uptake of arsenite into cells, rendering them sensitive to arsenite. To understand the role of aqpS in arsenic resistance, S. meliloti aqpS and arsC were disrupted individually. Disruption of aqpS resulted in increased tolerance to arsenite but not arsenate, while cells with an arsC disruption showed selective sensitivity to arsenate. The results of transport experiments in intact cells suggest that AqpS is the only protein of the S. meliloti ars operon that facilitates transport of arsenite. Coexpression of S. meliloti aqpS and arsC in a strain of E. coli lacking the ars operon complemented arsenate but not arsenite sensitivity. These results imply that, when S. meliloti is exposed to environmental arsenate, arsenate enters the cell through phosphate transport systems and is reduced to arsenite by ArsC. Internally generated arsenite flows out of the cell by downhill movement through AqpS. Thus, AqpS confers arsenate resistance together with ArsC-catalyzed reduction. This is the first report of an aquaglyceroporin with a physiological function in arsenic resistance

Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin

Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field

Common factors of antimonial resistance in different species of <i>Leishmania</i>.

<p><b>A</b>. Levels of <i>MRPA</i> mRNA: Total RNA was isolated from promastigotes of different species and <i>MRPA</i> mRNA expression levels were estimated using qPCR. Relative (with respect to <i>L</i>. <i>donovani</i>) MRPA expression levels were calculated using 2^-ΔΔCt method. Data were expressed as mean ± SD of three independent experiments in triplicate. <b>B</b>. Levels of total non-protein thiols: Promastigotes from different species of <i>Leishmania</i> were harvested and proteins were precipitated using tricloroacetic acid. Total non-protein thiols were estimated using dithionitrobenzoic acid. Data were expressed as mean ± SE of three independent experiments in triplicate. <b>C</b>. Levels of <i>AQP1</i> mRNA: Total RNA was isolated from promastigotes of different species and <i>AQP1</i> mRNA expression levels were estimated using qPCR. Relative (with respect to <i>L</i>. <i>donovani</i>) AQP1 expression levels were calculated using 2^-ΔΔCt method. Data were expressed as mean ± SD of three independent experiments in triplicate.</p