Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defence against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen‐activated protein kinase, LmjMPK2. Leishmania parasites coexpressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo‐osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr‐197 and this phosphorylation requires LmjMPK2 activity. Lys‐42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild‐type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. Leishmania mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild‐type cells. This is the first report where a parasite aquaglyceroporin activity is post‐translationally modulated by a mitogen‐activated protein kinase.     

Osteoclast differentiation and function in aquaglyceroporin AQP9-null mice

Background information. Osteoclasts are cells specialized for bone resorption and play important roles in bone growth and calcium homoeostasis. Differentiation of osteoclasts involves fusion of bone marrow macrophage mononuclear precursors in response to extracellular signals. A dramatic increase in osteoclast cell volume occurs during osteoclast biogenesis and is believed to be mediated by AQP9 (aquaporin 9), a membrane protein that can rapidly transport water and other small neutral solutes across cell membranes. Results. In the present study we report an increase in expression of AQP9 during differentiation of a mouse macrophage cell line into osteoclasts. Bone marrow macrophages from wild‐type and AQP9‐null mice differentiate into osteoclasts that have similar morphology, contain comparable numbers of nuclei, and digest synthetic bone to the same extent. Bones from wild‐type and AQP9‐null mice contain similar numbers of osteoclasts and have comparable density and structure as measured by X‐ray absorptiometry and microcomputed tomography. Conclusions. Our results confirm that AQP9 expression rises during osteoclast biogenesis, but indicate that AQP9 is not essential for osteoclast function or differentiation under normal physiological conditions.     

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.     

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.     

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.     

Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene

Aquaporin-1 (AQP1) is a water channel that is induced by hypertonicity. The present study was undertaken to clarify the osmoregulation mechanism of AQP1 in renal medullary cells. In cultured mouse medullary (mIMCD-3) cells, AQP1 expression was significantly induced by hypertonic treatment with impermeable solutes, whereas urea had no effect on AQP1 expression. This result indicates the requirement of a hypertonic gradient. Hypertonicity activated ERK, p38 kinase, and JNK in mIMCD-3 cells. Furthermore, all three MAPKs were phosphorylated by the upstream activation of MEK1/2, MKK3/6, and MKK4, respectively. The treatments with MEK inhibitor U0126, p38 kinase inhibitor SB203580, and JNK inhibitor SP600125 significantly attenuated hypertonicity-induced AQP1 expression in mIMCD-3 cells. In addition, hypertonicity-induced AQP1 expression was significantly reduced by both the dominant-negative mutants of JNK1- and JNK2-expressing mIMCD-3 cells. NaCl-inducible activity of AQP1 promoter, which contains a hypertonicity response element, was attenuated in the presence of U0126, SB203580, and SP600125 in a dose-dependent manner and was also significantly reduced by the dominant-negative mutants of JNK1 and JNK2. These data demonstrate that the activation of ERK, p38 kinase, and JNK pathways and the hypertonicity response element in the AQP1 promoter are involved in hypertonicity-induced AQP1 expression in mIMCD-3 cells.     

Effect of mitomycin C on specific gene transcript levels in cultured mammalian cells

The effect of mitomycin C on the accumulation of specific mRNAs was studied in asynchronously growing Swiss 3T3 cells, as well as in synchronously growing serum stimulated ts13 cells (a temperature-sensitive mutant from the BHK cell line). It was observed that the steady-state level of p53 RNA experienced some increase in 3T3 cells treated for 24 h with the drug. In addition, mitomycin when applied to serum stimulated ts13 cells increased the level of p2F1 RNA. Mitomycin diminished the level of core histone H3 RNA, a finding consistent with the inhibitory action of this compound on DNA replication.     

Selection for cold resistance alters gene transcript levels in Drosophila melanogaster

Microarrays have been used to examine changes in gene expression underlying responses to selection for increased stress resistance in Drosophila melanogaster, but changes in expression patterns associated with increased resistance to cold stress have not been previously reported. Here we describe such changes in basal expression levels in replicate lines following selection for increased resistance to chill coma stress. We found significant up- or down-regulation of expression in 94 genes on the Affymetrix Genome 2.0 array. Quantitative RT-PCR was used to confirm changes in expression of six genes. Some of the identified genes had previously been associated with stress resistance but no previously identified candidate genes for cold resistance showed altered patterns of expression. Seven differentially expressed genes that form a tight chromosomal cluster and an unlinked gene AnnX may be potentially important for cold adaptation in natural populations. Artificial selection for chill coma resistance therefore altered basal patterns of gene expression, but we failed to link these changes to plastic changes in expression under cold stress or to previously identified candidate genes for components of cold resistance.     

Alteration in glycerol and metalloid permeability by a single mutation in the extracellular C‐loop of Leishmania major aquaglyceroporin LmAQP1

The Leishmania major aquaglyceroporin, LmAQP1, is responsible for the transport of antimonite [Sb(III)], an activated form of Pentostam or Glucantime. Downregulation of LmAQP1 provides resistance to trivalent antimony compounds and increased expression of LmAQP1 in drug‐resistant parasites can reverse the resistance. Besides metalloid transport, LmAQP1 is also permeable to water, glycerol, methylglyoxal, dihydroxyacetone and sugar alcohols. LmAQP1 also plays a physiological role in volume regulation and osmotaxis. In this study, we examined the role of extracellular C‐loop glutamates (Glu143, Glu145 and Glu152) in LmAQP1 activity. Alteration of both Glu143 and Glu145 to alanines did not affect either the biochemical or physiological properties of the protein, suggesting that neither residue is critical for LmAQP1 activity. Alteration of Glu152 to alanine, aspartate and glutamine affected metalloid transport in the order, wild‐type > E152Q > E152D > E152A. In fact, axenic amastigotes expressing E152A LmAQP1 accumulated negligible levels of either arsenite [As(III)] or Sb(III). Alteration of Glu152 significantly affected volume regulation and osmotaxis, suggesting that Glu152 is critical for the physiological activity of the parasite. More importantly, alteration of Glu152 to alanine did not affect glycerol permeability. Although the metalloids, As(III) and Sb(III), are believed to be transported through aquaglyceroporin channels as they behave as inorganic molecular mimic of glycerol, this is the first report where metalloid and glycerol transport can be dissected by a single mutation at the extracellular pore entry of LmAQP1 channel.