Mammalian microRNA: an important modulator of host-pathogen interactions in human viral infections.

MicroRNAs (miRNAs), which are small non-coding RNAs expressed by almost all metazoans, have key roles in the regulation of cell differentiation, organism development and gene expression. Thousands of miRNAs regulating approximately 60 % of the total human genome have been identified. They regulate genetic expression either by direct cleavage or by translational repression of the target mRNAs recognized through partial complementary base pairing. The active and functional unit of miRNA is its complex with Argonaute proteins known as the microRNA-induced silencing complex (miRISC). De-regulated miRNA expression in the human cell may contribute to a diverse group of disorders including cancer, cardiovascular dysfunctions, liver damage, immunological dysfunction, metabolic syndromes and pathogenic infections. Current day studies have revealed that miRNAs are indeed a pivotal component of host-pathogen interactions and host immune responses toward microorganisms. miRNA is emerging as a tool for genetic study, therapeutic development and diagnosis for human pathogenic infections caused by viruses, bacteria, parasites and fungi. Many pathogens can exploit the host miRNA system for their own benefit such as surviving inside the host cell, replication, pathogenesis and bypassing some host immune barriers, while some express pathogen-encoded miRNA inside the host contributing to their replication, survival and/or latency. In this review, we discuss the role and significance of miRNA in relation to some pathogenic viruses.

Functional role of lysine 12 in Leishmania major AQP1.

Leishmania major aquaglyceroporin (AQP1) is an adventitious metalloid channel that allows the bidirectional movement of arsenite and antimonite. Here we demonstrate that AQP1 is subjected to proteasome-dependent degradation. Treatment of Leishmania promastigotes with the proteasome inhibitor MG132 resulted in increased AQP1 accumulation. Site-directed mutagenesis in AQP1 revealed that alteration of lysine 12 to either alanine or arginine improves protein stability. AQP1 expression is stabilized by mitogen-activated protein kinase 2 (MPK2). Cells expressing a dominant-negative MPK2 mutant exhibited severely reduced AQP1 expression, which could be reversed upon addition of MG132. Interestingly, the dominant-negative MPK2 mutant could not destabilize either AQP1K12A or AQP1K12R. While stabilization of AQP1 by MPK2 leads to its relocalization from flagellum to the entire surface of the parasite, altered AQP1K12A or AQP1K12R was restricted to flagellum only. Our data demonstrate that lysine 12 is targeted for proteasomal degradation of AQP1 and plays an integral role in subcellular localization of AQP1 as well as its interaction with MPK2. This work also raises the possibility that a strategy combining antimonial with a proteasome inhibitor may be an effective combination regimen against diverse forms of leishmaniasis.

Generation of an aquaglyceroporin AQP1 null mutant in Leishmania major.

The Leishmania aquaglyceroporin AQP1 plays an important physiological role in water and uncharged polar solutes transport, volume regulation, osmotaxis, and is a key determinant of antimony resistance. By targeted gene disruption, we generated a Leishmania major promastigote AQP1 null mutant. This required several attempts but a chromosomal null AQP1 mutant was obtained by loss of heterozygosity in the presence of a rescue plasmid encoding AQP1. Growth in the absence of selection led to the loss of the rescuing plasmid, indicating that AQP1 is not essential for Leishmania viability. The AQP1-null mutant was resistant to antimonyl tartrate (SbIII) and arsenite (AsIII) due to a decrease import of these metalloids. It also exhibited alterations in its osmoregulation abilities compared with wild-type cells. This is the first report of the generation of a genetic AQP1 null mutant in Leishmania parasite, confirming its physiological function and role in resistance to antimonials, the therapeutic mainstay against Leishmania.

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.

Aquaglyceroporins: generalized metalloid channels.

BACKGROUND: Aquaporins (AQPs), members of a superfamily of transmembrane channel proteins, are ubiquitous in all domains of life. They fall into a number of branches that can be functionally categorized into two major sub-groups: i) orthodox aquaporins, which are water-specific channels, and ii) aquaglyceroporins, which allow the transport of water, non-polar solutes, such as urea or glycerol, the reactive oxygen species hydrogen peroxide, and gases such as ammonia, carbon dioxide and nitric oxide and, as described in this review, metalloids. SCOPE OF REVIEW: This review summarizes the key findings that AQP channels conduct bidirectional movement of metalloids into and out of cells. MAJOR CONCLUSIONS: As(OH)3 and Sb(OH)3 behave as inorganic molecular mimics of glycerol, a property that allows their passage through AQP channels. Plant AQPs also allow the passage of boron and silicon as their hydroxyacids, boric acid (B(OH)3) and orthosilicic acid (Si(OH)4), respectively. Genetic analysis suggests that germanic acid (GeO2) is also a substrate. While As(III), Sb(III) and Ge(IV) are toxic metalloids, borate (B(III)) and silicate (Si(IV)) are essential elements in higher plants. GENERAL SIGNIFICANCE: The uptake of environmental metalloids by aquaporins provides an understanding of (i) how toxic elements such as arsenic enter the food chain; (ii) the delivery of arsenic and antimony containing drugs in the treatment of certain forms of leukemia and chemotherapy of diseases caused by pathogenic protozoa; and (iii) the possibility that food plants such as rice could be made safer by genetically modifying them to exclude arsenic while still accumulating boron and silicon. This article is part of a Special Issue entitled Aquaporins.

Trypanosoma brucei aquaglyceroporins facilitate the uptake of arsenite and antimonite in a pH dependent way.

BACKGROUND: Trypanosoma brucei is a primitive parasitic protozoan that thrives in diverse environments such as the midgut of the tsetse fly and the blood of a mammalian host. For an adequate adaptation to these environments, the parasite's aquaglyceroporins play an important role. METHODS AND RESULTS: In order to test their ability to transport trivalent arsenic and antimony, we expressed the three known Trypanosoma brucei aquaglyceroporins (TbAQPs) in the heterologous systems of yeast null aquaporin mutant and Xenopus laevis oocytes. For both expression systems, we found a pH dependent intracellular accumulation of As(III) or Sb(III) mediated by all of the three TbAQPs, with the exception of TbAQP1-As(III) uptake. Additionally, we observed that Trypanosoma brucei aquaglyceroporins allow the passage of As(III) in both directions. CONCLUSION: Taken together, these results demonstrated that T. brucei aquaglyceroporins can serve as entry routes for As(III) and Sb(III) into the parasitic cell, and that this uptake is pH sensitive. Therefore, aquaporins of protozoan parasites may be considered useful as a vehicle for drug delivery.

Telomeric gene deletion and intrachromosomal amplification in antimony-resistant Leishmania.

Antimonials are still the mainstay of treatment against leishmaniasis but drug resistance is increasing. We carried out short read next-generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony-resistant mutants. Copy number variations were consistently detected with both NGS and CGH. A major attribute of antimony resistance was a novel terminal deletion of variable length (67 kb to 204 kb) of the polyploid chromosome 31 in the three mutants. Terminal deletions in two mutants occurred at the level of inverted repeated sequences. The AQP1 gene coding for an aquaglyceroporin was part of the deleted region and its transfection into resistant mutants reverted resistance to SbIII. We also highlighted an intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded for ascorbate-dependent peroxidase (APX) and glucose-6-phosphate dehydrogenase (G6PDH). Overexpression of these genes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS). Generation of a G6PDH null mutant in one revertant exhibited SbIII sensitivity and a decreased protection of ROS. Our genomic analyses and functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania.

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.

The role of alanine 163 in solute permeability of Leishmania major aquaglyceroporin LmAQP1.

Leishmania major aquaglyceroporin LmAQP1 allows adventitious passage of antimonite, an activated form of the drug Pentostam, which is used as the first line treatment for leishmaniasis. The extracellular C-loop of an aquaglyceroporin confers substrate specificity. Alteration of Glu125 to serine in the Plasmodium falciparum aquaglyceroporin PfAQP has been shown to selectively affect water but not glycerol permeability. The C-loop of LmAQP1 is twelve residues longer than PfAQP, and Ala163 is at an equivalent position as Glu125 of PfAQP. The role of Ala163 in LmAQP1 solute permeability was investigated. Alteration of Ala163 to serine or threonine did not significantly affect conduction of solutes. However, alteration to aspartate, glutamate, and glutamine blocked passage of water, glycerol, and other organic solutes. While LmAQP1 is a mercurial insensitive water channel, mutation of the adjacent threonine (Thr164) to cysteine led to inhibition of water passage by Hg(2+). This inhibition could be reversed upon addition of ß-mercaptoethanol. These data suggest that, unlike Glu125 (PfAQP), Ala163 is not involved in stabilization of the C-loop and selective solute permeability. Ala163 is located near the pore mouth of the channel, and replacement of Ala163 by bulkier residue sterically hinders the passage of solutes. Alteration of Ala163 to serine or threonine affected metalloid uptake in the order, wild-type>A163S>A163T. Metalloid conduction was near completely blocked when Ala163 was mutagenized to aspartate, glutamate, or glutamine. Mutations such as A163S and A163T that reduced the permeability to antimonite, without a significant loss in water or solute conductivity raises the possibility that, subtle changes in the side chain of the amino acid residue in position 163 of LmAQP1 may play a role in drug resistance.

Metalloid transport by aquaglyceroporins: consequences in the treatment of human diseases.

Metalloids can severely harm human physiology in a toxicological sense if taken up from the environment in acute high doses or chronically. However, arsenic or antimony containing drugs are still being used as treatment and are often the sole regime for certain forms of cancer, mainly types of leukemia and diseases caused by parasites, such as sleeping sickness or leishmaniasis. In this chapter, we give an outline of the positive effects of arsenicals and antimonials against such diseases, we summarize data on uptake pathways through human and parasite aquaglyceroporins and we discuss the progress and options in the development of therapeutic aquaporin and aquaglyceroporin inhibitor compounds.

Adventitious arsenate reductase activity of the catalytic domain of the human Cdc25B and Cdc25C phosphatases.

A number of eukaryotic enzymes that function as arsenate reductases are homologues of the catalytic domain of the human Cdc25 phosphatase. For example, the Leishmania major enzyme LmACR2 is both a phosphatase and an arsenate reductase, and its structure bears similarity to the structure of the catalytic domain of human Cdc25 phosphatase. These reductases contain an active site C-X(5)-R signature motif, where C is the catalytic cysteine, the five X residues form a phosphate binding loop, and R is a highly conserved arginine, which is also present in human Cdc25 phosphatases. We therefore investigated the possibility that the three human Cdc25 isoforms might have adventitious arsenate reductase activity. The sequences for the catalytic domains of Cdc25A, -B, and -C were cloned individually into a prokaryotic expression vector, and their gene products were purified from a bacterial host using nickel affinity chromatography. While each of the three Cdc25 catalytic domains exhibited phosphatase activity, arsenate reductase activity was observed only with Cdc25B and -C. These two enzymes reduced inorganic arsenate but not methylated pentavalent arsenicals. Alteration of either the cysteine and arginine residues of the Cys-X(5)-Arg motif led to the loss of both reductase and phosphatase activities. Our observations suggest that Cdc25B and -C may adventitiously reduce arsenate to the more toxic arsenite and may also provide a framework for identifying other human protein tyrosine phosphatases containing the active site Cys-X(5)-Arg loop that might moonlight as arsenate reductases.

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 NaAsO(2), AQP9-null mice suffer reduced survival rates (LD(50), 12 mg/kg) compared with WT mice (LD(50), 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 NaAsO(2) 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 approximately 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.

Structural characterization of the As/Sb reductase LmACR2 from Leishmania major.

The arsenate/antimonate reductase LmACR2 has been recently identified in the genome of Leishmania major. Besides displaying phosphatase activity in vitro, this enzyme is able to reduce both As(V) and Sb(V) to their respective trivalent forms and is involved in the activation of Pentostan, a drug containing Sb(V) used in the treatment of leishmaniasis. LmACR2 displays sequence and functional similarity with the arsenate reductase ScACR2 from Saccharomyces cerevisiae, and both proteins are homologous to the catalytic domain of Cdc25 phosphatases, which, in turn, belong to the rhodanese/Cdc25 phosphatase superfamily. In this work, the three-dimensional structure of LmACR2 has been determined with crystallographic methods and refined at 2.15 A resolution. The protein structure maintains the overall rhodanese fold, but substantial modifications are observed in secondary structure position and length. However, the conformation of the active-site loop and the position of the catalytic residue Cys75 are unchanged with respect to the Cdc25 phosphatases. From an evolutionary viewpoint, LmACR2 and the related arsenate reductases form, together with the known Cdc25 phosphatases, a well-defined subfamily of the rhodanese/Cdc25 phosphatase superfamily, characterized by a 7-amino-acid-long active-site loop that is able to selectively bind substrates containing phosphorous, arsenic, or antinomy. The evolutionary tree obtained for these proteins shows that, besides the active-site motif CE[F/Y]SXXR that characterizes Cdc25 phosphatase, the novel CALSQ[Q/V]R motif is also conserved in sequences from fungi and plants. Similar to Cdc25 phosphatase, these proteins are likely involved in cell cycle control. The active-site composition of LmACR2 (CAQSLVR) does not belong to either group, but gives to the enzyme a bifunctional activity of both phosphatase and As/Sb reductase. The subtle dependence of substrate specificity on the amino acid composition of the active-site loop displays the versatility of the ubiquitous rhodanese domain.

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.

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.

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.

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.

A CDC25 homologue from rice functions as an arsenate reductase.

Enzymatic reduction of arsenate to arsenite is the first step in arsenate metabolism in all organisms studied. The rice genome contains two ACR2-like genes, OsACR2.1 and OsACR2.2, which may be involved in regulating arsenic metabolism in rice. Here, we cloned both OsACR2 genes and expressed them in an Escherichia coli strain in which the arsC gene was deleted and in a yeast (Saccharomyces cerevisiae) strain with a disrupted ACR2 gene. OsACR2.1 complemented the arsenate hypersensitive phenotype of E. coli and yeast. OsACR2.2 showed much less ability to complement. The gene products were purified and demonstrated to reduce arsenate to arsenite in vitro, and both exhibited phosphatase activity. In agreement with the complementation results, OsACR2.1 exhibited higher reductase activity than OsACR2.2. Mutagenesis of cysteine residues in the putative active site HC(X)(5)R motif led to nearly complete loss of both phosphatase and arsenate reductase activities. In planta expression of OsACR2.1 increased dramatically after exposure to arsenate. OsACR2.2 was observed only in roots following arsenate exposure, and its expression was less than OsACR2.1.

Crystallization and preliminary crystallographic characterization of LmACR2, an arsenate/antimonate reductase from Leishmania major.

Arsenic is present in the biosphere owing either to the presence of pesticides and herbicides used in agricultural and industrial activities or to leaching from geological formations. The health effects of prolonged exposure to arsenic can be devastating and may lead to various forms of cancer. Antimony(V), which is chemically very similar to arsenic, is used instead in the treatment of leishmaniasis, an infection caused by the protozoan parasite Leishmania sp.; the reduction of pentavalent antimony contained in the drug Pentostam to the active trivalent form arises from the presence in the Leishmania genome of a gene, LmACR2, coding for the protein LmACR2 (14.5 kDa, 127 amino acids) that displays weak but significant sequence similarity to the catalytic domain of Cdc25 phosphatase and to rhodanese enzymes. For structural characterization, LmACR2 was overexpressed, purified to homogeneity and crystallized in a trigonal space group (P321 or P3(1)21/P3(2)21). The protein crystallized in two distinct trigonal crystal forms, with unit-cell parameters a = b = 111.0, c = 86.1 A and a = b = 111.0, c = 175.6 A, respectively. At a synchrotron beamline, the diffraction pattern extended to a resolution limit of 1.99 A.

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.