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Alteration in glycerol and metalloid permeability by a single mutation in the extracellular C-loop of Leishmania major aquaglyceroporin LmAQP1
Abstract
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.
... Aquaglyceroporin 1 in Leishmania parasites is associated with antimony resistance and several independent studies have revealed an evolutionary convergence leading to loss of function AQP1 mutations (insertion, deletion and SNPs) that impair SbIII uptake in both laboratory-selected and clinical isolates Leishmania spp. mutants [4,5,[22][23][24]. However, despite of characterizing the Indels and/or SNPs detection, none of the above mentioned works depicted the molecular dynamic alterations in AQP1 upon mutation acquisition. ...
... Alignment and topology were respectively performed using TEXshade and TEXtopo packages (Beitz 2000). Dark blue: conserved residues in Leishmania spp.; light blue: similar residues among Leishmania species; red highlighted circle: the site of Gly 133 Asp mutation in L. guyanensis; green highlighted circle: residues previously associated with antimony resistance in Leishmania major [7,21,22]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) introduction of a charged residue, aspartic acid, an alteration that plays with both, monomer and tetramer-formation. ...
... Compared with L. guyanensis AQP1 protein, G562A mutation in L. major (position 189) correspond to the residue Serine 160 from Viannia subgenus sequence and is conserved among Leishmania parasites, located at the extracellular C-loop C of AQP1 topology (Fig. 1). This region was previously associated with impaired metalloid uptake and reduced antimony permeability [21,22]. However, these previous reports are based on induced mutations. ...
The Leishmania aquaglyceroporin 1 (AQP1) plays an important role in osmoregulation and antimony (Sb) uptake, being determinant for resistance to antimony. We have previously demonstrated that G133D mutation on L. guyanensis AQP1 (LgAQP1) leads to reduced Sb uptake. Here, we investigated the effects of G133D mutation on LgAQP1 structure, associated with Sb uptake and alterations in osmoregulation capacity. High confidence molecular models of wild-type LgAQP1 as well as the LgAQP1::G133D mutant were constructed and optimized via comparative homology modeling. Computational methods from the mCSM platform were used to evaluate the effects on protein stability and on its ability to bind to glycerol. Functional validation of the disruptive effect of the mutation on LgAQP1 was done by challenging the parasites with hypo-osmotic chock. Glycine 133 is on transmembrane helix 3, buried in the membrane in both open and closed conformation. G133D mutation was predicted to be highly destabilizing, as it alters the helical bundling arrangement in order to accommodate the aspartic acid side chain. The shift in helices also resulted in fewer favorable contacts with glycerol in the channel, which would explain the reduced affinity for similar small molecules as SbO3. Under hypo-osmotic condition, L. guyanensis AQP1G133D presented a 3-fold increase in cellular volume and pronounced delay to recover osmosis homeostasis when compared to the wild-type, a profile that was enhanced in LgAQP1-/- mutants. In conclusion, G133D is a highly disruptive mutation that will destabilize the monomer, compromise tetramer formation and alter pore conformation, leading to reduced Sb uptake and deficient osmoregulation.
... among others [15][16][17][18] . However, the use of a unique system for functional expression of aquaglyceroporins may face a number of constraints. ...
... The values obtained over these days were constant and compatible with simple membrane diffusion (between 15.4 and 17.9 μm.s −1 ). Interestingly, these values are comparable to those reported in the literature for X. laevis oocytes 13,17 . Expression of hAQP1 led to a very fast cell swelling as compared to control oocytes (Fig. 2a). ...
... Under these conditions, a glycerol gradient is created maintaining the iso-osmolarity. The influx of glycerol across the aquaglyceroporin into the oocytes changes the inside osmolarity of the cells and produces a concomitant water entry, leading to the oocyte's swelling proportionally to its glycerol permeability 16,17,26,30,38 . As shown in Fig. 3, when oocytes injected with 10 ng of TbAQP2-cRNA or TbAQP3-cRNA were subjected to the referred glycerol gradient, the association between swelling versus time displayed a linear behaviour with R 2 values close to 1 (Fig. 3), similar as it occurred for water permeability measurements by hypo-osmotic swelling assay (Fig. 1). ...
Amphibian oocytes have been extensively used for heterologous expression of membrane proteins for studying their biochemical and biophysical properties. So far, Xenopus laevis is the main amphibian used as oocytes source to express aquaglyceroporins in order to assess water and solutes permeability. However, this well-established amphibian model represents a threat to the biodiversity in many countries, especially in those from tropical regions. For that reason, the import of Xenopus laevis is subjected to strict control, which essentially has restricted its use in these regions. Therefore, a wider variety of expression systems for aquaglyceroporins is needed. Rhinella marina is extensively distributed in the Americas and its native range spreads from South America to Texas, US. Here we report the use of Rhinella marina oocytes as an alternative expression system for aquaglyceroporins and demonstrated its suitability to determine the permeability to water and non-ionic solutes. Rhinella marina oocytes were able to functionally express channels from human and the protozoan pathogen Trypanosoma brucei, two very distant organisms on the evolutionary scale. Permeability values obtained from Rhinella marina oocytes expressing members of aquaporin family were similar and comparable to those values reported in the literature for the same channels expressed in Xenopus laevis oocytes.
... While the structure of L. major AQP1 is yet to be elucidated, molecular modeling based on the three-dimensional structure of Plasmodium falciparum AQP (PfAQP) (9), along with structure-function correlations of L. major AQP1 have defined the residues lining the outer mouth of the channel (Fig. S1). Glutamate 152 on the second extracellular loop near the pore mouth helps the channel differentiate between antimonite and glycerol (10). Mutations in alanine 163 in the same loop altered the permeability to antimonite without any significant loss in water or solute conductivity (11). ...
... LdBob cells were grown as promastigotes as described earlier (8,13). While an AQP1 null-mutant in L. major has only recently been developed at Ouellette laboratory (15), the LdBob cell line was chosen based on a logical extension of our previous studies (7,8,10,11,13). Also, LdBob cells have the lowest AQP1 mRNA levels, are highly resistant to Sb(III), show lowest Sb(III) accumulation, and exhibit poor osmoregulatory activity (7), which makes LdBob as good as an AQP1 knockdown strain. ...
... AQP1 shares 32% identity and 50% similarity with Plasmodium falciparum aquaglyceroporin (PfAQP). A topology prediction of AQP1 based on the crystal structure of PfAQP (9) predicted the AQP1 N-and C-termini to be intracellular (Fig. S1) (10). Similarly, the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) ...
... Point mutations in the AQP-1 gene could give rise to clinical drug resistance [16]. We observed two missense SNPs in AQP-1 gene out of the 84 SNPs detected, to be associated with drug resistance phenotype [30,31]. At the position, A188 Alanine was replaced by Threonine (A188T). ...
... This change has been reported not to affect the 3D structure of the protein but shown to be susceptible to post-translational modification, which may affect the protein's function, thus altering the uptake of antimonials [31]. A study conducted in Leishmania major AQP-11 showed a missense SNP at E152 (corresponding to E185 in the full-length sequence), which conferred Sb III resistance to laboratory mutants of the parasite [30]. This mutation corresponds to the observed SNP in our study at E185 (E185A) full-length coding sequence (945 bp) of L. donovani. ...
Visceral Leishmaniasis (VL) remains a major public health problem mainly affecting the poorest populations across Asia, Africa, Middle East, Europe, Southern and Central America. For seven-decade now, the first-line drug of choice for leishmaniasis has been pentavalent antimonials. However, the clinical value of these drugs is threatened by the emergence of drug-resistant parasites. Clinical resistance to sodium stibogluconate (pentostam) has been a challenge in the Indian subcontinent, raising concerns for the endemic countries in Africa. This study aimed to identify and describe Single Nucleotide Polymorphism (SNPs) in gene markers associated with drug resistance among the clinical samples. The study was an experimental laboratory investigation on Dry Blood Spots (DBS). DNA was extracted from 18 VL positive samples, and Internal Transcribed Spacer-1 Polymerase Chain Reaction confirmed the positivity. Two target resistance markers, aquaglyceroporin 1 (AQP-1) and the Multi-Drug Resistant Protein A (MRPA), were PCR-amplified and resulting amplicons sequenced using the Sanger sequencing platform. Multiple sequence alignments were performed using ClustalW, and the phylogenetic tree was constructed in MegaX using the Maximum Likelihood method. A total of 84 SNPs in the AQP-1 gene were identified from six clinical samples. Fifty-nine of the SNPs (70.2%) were non-synonymous, while 25 (29.8%) were synonymous. Among the non-synonymous SNPs, three (5.1%) were nonsense, and 56 (94.9%) were missense point mutations. Two missense SNPs A188T and E185A in S17608 reported to be associated with drug resistance phenotype were observed. The study describes the resistance associated with the pentostam uptake by Leishmania donovani.
... Loop C of different NIP4s varies by up to two amino acids in length (Figure 7). These variations in sequence length and amino acid composition (Supplementary File S5) are of interest, as loop C was shown to be involved in the substrate selectivity of various AQPs (Beitz et al., 2004;Uzcategui et al., 2008). A single mutation in the extracellular loop C of Leishmania AQP1 resulted in altered substrate selectivity preventing metalloid but not glycerol permeation through the channel (Uzcategui et al., 2008). ...
... These variations in sequence length and amino acid composition (Supplementary File S5) are of interest, as loop C was shown to be involved in the substrate selectivity of various AQPs (Beitz et al., 2004;Uzcategui et al., 2008). A single mutation in the extracellular loop C of Leishmania AQP1 resulted in altered substrate selectivity preventing metalloid but not glycerol permeation through the channel (Uzcategui et al., 2008). The overall similarity between AQPs from Arabidopsis, B. oleracea and B. rapa at the protein level, and especially at the residues constituting the NPA and ar/R selectivity filters, suggests that the knowledge on channel selectivity which was revealed in Arabidopsis might be transferable to Brassica crop isoforms. ...
Aquaporins (AQPs) are essential channel proteins that regulate plant water homeostasis and the uptake and distribution of uncharged solutes such as metalloids, urea, ammonia, and carbon dioxide. Despite their importance as crop plants, little is known about AQP gene and protein function in cabbage (Brassica oleracea) and other Brassica species. The recent releases of the genome sequences of B. oleracea and Brassica rapa allow comparative genomic studies in these species to investigate the evolution and features of Brassica genes and proteins. In this study, we identified all AQP genes in B. oleracea by a genome-wide survey. In total, 67 genes of four plant AQP subfamilies were identified. Their full-length gene sequences and locations on chromosomes and scaffolds were manually curated. The identification of six additional full-length AQP sequences in the B. rapa genome added to the recently published AQP protein family of this species. A phylogenetic analysis of AQPs of Arabidopsis thaliana, B. oleracea, B. rapa allowed us to follow AQP evolution in closely related species and to systematically classify and (re-) name these isoforms. Thirty-three groups of AQP-orthologous genes were identified between B. oleracea and Arabidopsis and their expression was analyzed in different organs. The two selectivity filters, gene structure and coding sequences were highly conserved within each AQP subfamily while sequence variations in some introns and untranslated regions were frequent. These data suggest a similar substrate selectivity and function of Brassica AQPs compared to Arabidopsis orthologs. The comparative analyses of all AQP subfamilies in three Brassicaceae species give initial insights into AQP evolution in these taxa. Based on the genome-wide AQP identification in B. oleracea and the sequence analysis and reprocessing of Brassica AQP information, our dataset provides a sequence resource for further investigations of the physiological and molecular functions of Brassica crop AQPs.
... AQP1 is considered the major route of entry of trivalent antimony in Leishmania [27] and its overexpression leads to Sb III hypersensitivity [26,27]. Downregulation of AQP1 has been observed in both laboratory-raised and clinical Leishmania parasites resistant to antimony [26,31,67,68] and constitutes a potentially [71] and PfAQP [77,78]. Color-code consensus is based on the alignment presented in panel B. The single mutation G133D is indicated in transmembrane domain III and is conserved in all organisms. ...
... This prompted us to sequence AQP1 and to demonstrate for the first time that a point mutation in AQP1 (G133D) can also be a novel resistance mechanism. Mutational analysis on LmAQP1 had already revealed that residues located at C-loop Ala163 and Glu152 (equivalent to LgAQP1 residues Val167 and Glu156, respectively- Fig. 8A) are involved in metalloid uptake and reduced permeability to antimony [28,71]. LmAQP1 is also post-transcriptionally regulated by a mitogen activated protein kinase 2-mediated phosphorylation at Thr197 (LgAQP1 Thr201- Fig. 8A) modulating Sb III uptake and sensitivity [72]. ...
Antimony resistance complicates the treatment of infections caused by the parasite Leishmania.
Using next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR) mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1). Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion.
This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.
... Although no crystal structure of LmAQP1 has been determined, structure-function studies have defined the pore mouth residues of LmAQP1 [84]. An extracellular loop near the Cterminus that includes residue Glu152 helps the channel to differentiate between metalloids and glycerol. ...
... Leishmania LmAQP1 has an extracellular loop with several glutamate residues. A single E152A mutation reduced uptake of As(III) and Sb(III) to negligible levels without affecting glycerol permeability [84], demonstrating that there is considerable plasticity in the selectivity of the aquaglyceroporin pore. While engineering human genes is less realistic, identification of drugs or pharmacological agents that reduce uptake of As(III) by AQP9 in liver might lead to amelioration of arsenic-related diseases such as bladder cancer. ...
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.
This review summarizes the key findings that AQP channels conduct bidirectional movement of metalloids into and out of cells.
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.
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.
... Despite the information provided by the crystal structures of GlpF and PfAQP, the contribution of individual residues in the conduction channel to solute permeability is not yet fully understood. We have undertaken site directed mutagenesis studies to gain insights into the solute selectivity of LmAQP1 and have earlier shown that alteration of Glu152 of LmAQP1 to alanine selectively abrogates metalloid but not glycerol permeability [10]. The objective of this work was to examine the role of Ala163 in solute permeability of LmAQP1. ...
... An in-depth understanding of the extracellular pore mouth and delineation of the subtle differences in channel properties of LmAQP1 from that of human AQPs would lead to the development of small molecule inhibitors that can selectively target protozoan AQPs. We have previously shown that alteration of Glu152 to alanine selectively abrogates metalloid but not glycerol permeability [10]. Identification of other residues that play a role in water or solute conductance as well as metalloid permeability is currently in progress. ...
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 β-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.
... Moreover, MRPA and AQP1 are involved in Sb uptake and their downregulation causes drug resistance both in vivo and in vitro. Thus, these Sb resistances may serve as a marker for the treatment of SNPs based on plasticity on a diverse set of isolates (Gourbal et al., 2004;Decuypere et al., 2005;Marquis et al., 2005;Uzcategui et al., 2008;Mandal et al., 2010;Mandal et al., 2015;Monte-Neto et al., 2015;Plourde et al., 2015). ...
Leishmania is an intracellular, zoonotic, kinetoplastid eukaryote with more than 1.2 million cases all over the world. The leishmanial chromosomes are divided into polymorphic chromosomal ends, conserved central domains, and antigen-encoding genes found in telomere-proximal regions. The genome flexibility of chromosomal ends of the leishmanial parasite is known to cause drug resistance and intracellular survival through the evasion of host defense mechanisms. Therefore, in this review, we discuss the plasticity of Leishmania genome organization which is the primary cause of drug resistance and parasite survival. Moreover, we have not only elucidated the causes of such genome plasticity which includes aneuploidy, epigenetic factors, copy number variation (CNV), and post-translation modification (PTM) but also highlighted their impact on drug resistance and parasite survival.
... Increased copy number of MAPK1 is thus associated with isolates from geographical locations with high historical antimonial drug pressures such as the Indian subcontinent and to a lesser extend Africa. Another protein, the membrane channel protein aquaglyceroporin (AQP1), is known to be involved in the uptake of pentavalent antimonials: reduced copy number and expression have been associated with drug resistance (Andrade et al., 2016;Gourbal et al., 2004;Monte-Neto et al., 2015;Mukherjee et al., 2013), as has other genetic variation at this locus Monte-Neto et al., 2015;Uzcategui et al., 2008). In our dataset, copy number at this locus was reduced in 6% and increased in 35% of all isolates with small effect sizes (CN À2 to À1 and +1 to +3) but at least one copy of the locus was always present (Figure 9-figure supplement 2B, Table 2). ...
Protozoan parasites of the Leishmania donovani complex – L. donovani and L. infantum – cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution. L. donovani isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of L. infantum samples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of the Leishmania donovani complex.
... Increased copy number of MAPK1 is thus associated with isolates from geographical locations with high historical antimonial drug pressures such as the Indian subcontinent and to a lesser extend Africa. Another protein, the membrane channel protein aquaglyceroporin (AQP1), is known to be involved in the uptake of pentavalent antimonials: reduced copy number and expression have been associated with drug resistance (Andrade et al., 2016;Gourbal et al., 2004;Monte-Neto et al., 2015;Mukherjee et al., 2013), as has other genetic variation at this locus Monte-Neto et al., 2015;Uzcategui et al., 2008). In our dataset, copy number at this locus was reduced in 6% and increased in 35% of all isolates with small effect sizes (CN À2 to À1 and +1 to +3) but at least one copy of the locus was always present (Figure 9-figure supplement 2B, Table 2). ...
Protozoan parasites of the Leishmania donovani complex – L. donovani and L. infantum – cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution. L. donovani isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of L. infantum samples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of the Leishmania donovani complex.
... Increased copy number of MAPK1 is thus associated with isolates from geographical locations with high historical antimonial drug pressures such as the Indian subcontinent and to a lesser extend Africa. Another protein, the membrane channel protein aquaglyceroporin (AQP1), is known to be involved in the uptake of pentavalent antimonials: reduced copy number and expression have been associated with drug resistance (Andrade et al., 2016;Gourbal et al., 2004;Monte-Neto et al., 2015;Mukherjee et al., 2013), as has other genetic variation at this locus Monte-Neto et al., 2015;Uzcategui et al., 2008). In our dataset, copy number at this locus was reduced in 6% and increased in 35% of all isolates with small effect sizes (CN À2 to À1 and +1 to +3) but at least one copy of the locus was always present (Figure 9-figure supplement 2B, Table 2). ...
Protozoan parasites of the Leishmania donovani complex – L. donovani and L. infantum – cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution. L. donovani isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of L. infantum samples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of the Leishmania donovani complex.
... G133D in AQP1 can be one of the mechanisms in non-healing isolates of Leishmania. Moreover, some other mutational analyses have been performed on LmAQP1 that revealed Ala163 and Glu152 are involved in metalloid uptake and reduced permeability to antimony (19,20). Therefore, in this study, we discovered the new resistance mechanism corresponding to a point mutation of AQP1. ...
Background: Regarding the antimonial-resistant of Leishmania spp., understanding of related mechanism is necessary. One of the most important involved molecules is aquaglyceropin1 (AQP1). The aim of this study was molecular analysis of AQP1 gene from antimonial-resistant clinical isolates and its expression.
Methods: Overall, 150 patients with cutaneous leishmaniasis referring to the reference laboratories of Yazd and Varzaneh,, located 105km southeast of Isfahan and 240km away from Yazd, were assessed from Jun 2015 to Dec 2017. After sampling, staining was done and evaluated for Leishman by microscope. Samples were collected in RNAlater solution for gene expression analysis in non-healing isolates. DNA extraction was performed from each slide with Leishman body. All patients with L. major isolates detected by ITS1-PCR-RFLP were followed for finding the resistant isolates, consequence of molecular characterization of AQP1 using PCR-RFLP. Gene expression of AQP1 from all resistant isolates was assessed in comparison with the one in a sensitive isolate. Statistical analysis was done using SPSS. The significance level was considered ≤0.05.
Results: Five isolates were detected as antimonial resistant. Molecular detection and identification were appeared that all were L. major. The molecular characterization of AQP1 showed G562A mutation. Gene expression of AQP1 in resistant isolates showed 1.67 fold higher than the sensitive isolate.
Conclusion: We reported a new point mutation of G562A in AQP1 gene involved in molecular mechanism in resistant isolates.
... This permeability is very high even if compared with others protozoa. Aquaglyceroporins from Leishmania (L.) major let pass methylglyoxal as well, but only at 55-70%, [36,37], compared to the rate of TbAQP2. It is important to emphasize that methylglyoxal permeability is not a general characteristic of AQPs, as other aquaglyceroporins do not allow passage of this metabolite, e.g., human AQP3 [38]. ...
Bloodstream forms of Trypanosoma (T.) brucei, the causative agent of African sleeping sickness, possess a highly active glycolysis, which generates as main end-products: pyruvate under aerobic conditions, and pyruvate and glycerol under anaerobic conditions. To secrete them into the extracellular milieu, the parasites have at least two main specific membrane proteins, the pyruvate transporter and the aquaglyceroporins However, there are several other minor products from the glycolysis that must be excreted by the parasites and whose exit pathway until now remained elusive. As aquaglyceroporins from T. brucei (TbAQP1, 2, and 3) show a wide permeability profile for small solutes, we decided to evaluate if these proteins allow the passage of methylglyoxal, L-lactate, D-lactate and acetate molecules. We expressed heterologously TbAQP1, 2, and 3 in aquaglyceroporin-null yeast cells or in Xenopus laevis oocytes and demonstrated that these channels are permeable for methylglyoxal, L-lactate, D-lactate and acetate. We further demonstrate that methylglyoxal is highly toxic for bloodstream forms of T. brucei, while L-lactate and D-lactate appear almost harmless. Additionally, we discuss all our findings in the light of the novel metabolic discoveries, putting in context the participation of TbAQP1, 2, 3, and other proteins in the excretion of unwanted metabolic end-products.
... While recent antimonial drugs such as SSG are compounds of pentavalent antimony (Sb V ), Sb V is thought to act mostly as a prodrug, being reduced to Sb III in both the macrophage phagolysosome (Frézard et al., 2001) and in the parasite itself (Denton et al., 2004;Decuypere et al., 2012). AQP1 is known to assist with Sb III uptake, both genetic and transcriptional changes at this locus have been associated with Sb resistance (Gourbal et al., 2004;Monte-Neto et al., 2015;Mukherjee et al., 2013;Uzcategui et al., 2008) and a homologous transporter is associated with drug resistance in trypanosomes (Baker et al., 2012). Recently, an AQP1 knockout line of Leishmania major was shown to be resistant to Sb III due to reduced uptake (Plourde et al., 2015). ...
ELife digest
The parasite Leishmania donovani causes a disease called visceral leishmaniasis that affects many of the world's poorest people. Around half a million new cases develop every year, but health authorities lack safe and effective drugs to treat them. Up to 80% of these cases occur in the Indian subcontinent, where devastating epidemics have occurred in the last decades.
One reason these epidemics continue to occur is that the parasites develop genetic mutations allowing them to adapt to and resist the drugs used to kill them. As there are few existing drugs that can kill L. donovani, it is crucial to understand how drug resistance emerges and spreads among parasite populations.
Imamura, Downing, Van den Broeck et al. have now investigated the history of visceral leishmaniasis epidemics by characterising the complete genetic sequence – or genome – of 204 L. donovani parasite samples. This revealed that the majority of parasites in the Indian subcontinent first appeared in the nineteenth century, matching the first historical records of visceral leishmaniasis epidemics.
The genomes show that most of the parasites are genetically similar and can be clustered into several closely related groups. These groups first appeared in the 1960s following the end of a regional campaign to eradicate malaria. The most common parasite group is particularly resistant to drugs called antimonials, which were the main treatment for leishmaniasis until recently. These parasites have a small genetic change that scrambles most of a protein known to be involved in the uptake of antimonials.
Parasites may also be able to develop resistance to drugs through additional mechanisms that allow them to produce many copies of the same gene. These mechanisms could allow the parasites to rapidly adapt to new drugs or changes in the populations it infects. The work of Imamura et al. looks only at parasites isolated from patients then grown in the laboratory, so further research is now needed to explore how variable the Leishmania genome is in both of the parasite’s hosts: humans and sandflies.
Imamura et al.’s study reveals how L. donovani has spread throughout the Indian subcontinent in fine detail. The genome data can be used to create simple molecular tools that could form an "early warning system" to track the success of disease control programs and to determine how well the current drugs are working.
DOI: http://dx.doi.org/10.7554/eLife.12613.002
... While recent antimonial drugs such as SSG are compounds of pentavalent antimony (Sb V ), Sb V is thought to act mostly as a prodrug, being reduced to Sb III in both the macrophage phagolysosome (Frézard et al., 2001) and in the parasite itself (Denton et al., 2004;Decuypere et al., 2012). AQP1 is known to assist with Sb III uptake, both genetic and transcriptional changes at this locus have been associated with Sb resistance (Gourbal et al., 2004;Monte-Neto et al., 2015;Mukherjee et al., 2013;Uzcategui et al., 2008) and a homologous transporter is associated with drug resistance in trypanosomes (Baker et al., 2012). Recently, an AQP1 knockout line of Leishmania major was shown to be resistant to Sb III due to reduced uptake (Plourde et al., 2015). ...
Leishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector-borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment.
... While recent antimonial drugs such as SSG are compounds of pentavalent antimony (Sb V ), Sb V is thought to act mostly as a prodrug, being reduced to Sb III in both the macrophage phagolysosome (Frézard et al., 2001) and in the parasite itself (Denton et al., 2004;Decuypere et al., 2012). AQP1 is known to assist with Sb III uptake, both genetic and transcriptional changes at this locus have been associated with Sb resistance (Gourbal et al., 2004;Monte-Neto et al., 2015;Mukherjee et al., 2013;Uzcategui et al., 2008) and a homologous transporter is associated with drug resistance in trypanosomes (Baker et al., 2012). Recently, an AQP1 knockout line of Leishmania major was shown to be resistant to Sb III due to reduced uptake (Plourde et al., 2015). ...
The parasite Leishmania donovani causes a fatal disease termed visceral leishmaniasis. The process through which the parasite adapts to environmental change remains largely unknown. Here we show that aneuploidy is integral for parasite adaptation and that karyotypic fluctuations allow for selection of beneficial haplotypes, which impact transcriptomic output and correlate with phenotypic variations in proliferation and infectivity. To avoid loss of diversity following karyotype and haplotype selection, L. donovani utilizes two mechanisms: polyclonal selection of beneficial haplotypes to create coexisting subpopulations that preserve the original diversity, and generation of new diversity as aneuploidy-prone chromosomes tolerate higher mutation rates. Our results reveal high aneuploidy turnover and haplotype selection as a unique evolutionary adaptation mechanism that L. donovani uses to preserve genetic diversity under strong selection. This unexplored process may function in other human diseases, including fungal infection and cancer, and stimulate innovative treatment options.
... This supports our hypothesis that the amount of AQP1 in the plasma membrane is the ratelimiting step in the uptake of the activated form of the antimonial drugs. A single-point mutation of Glu152 of AQP1 abolished the metalloid conductance without affecting the physiological functions of AQP1 [41]. Another point mutation at Ala163 also inhibited metalloid accumulation [42]. ...
Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. Pentavalent antimonials are currently the first line of defense for the treatment of both visceral (VL) and cutaneous leishmaniasis (CL). However, clinical resistance to this class of drug is a major impediment to treatment. Amphotericin B, pentamidine, and miltefosine offer significant promise in the treatment of VL and CL, including antimony-resistant cases. Several other drugs, for example, allopurinol, atovaquone, fluconazole, paromomycin, and sitamaquine, are in various stages of clinical trials. Application of many of these drugs has resulted in the development of either clinical- or laboratory-induced drug resistance. Understanding the mechanisms of resistance is important for the development of newer generation of drugs to provide better treatment of the disease.
... On the other hand, existing evidences suggest that occurrence of some special mutations could affect the gene expression level or efficiency of LmAQP1. Specifically, mutation of Glu152 and Arg230, located at C-loop of LmAQP1 could affect its expression [23][24] . It has been shown that mutations play an important role in changing phenotypes and pathogenicity [25][26] . ...
Background & objectives:
The mechanism of antimony resistance in Leishmania has been studied extensively, in connection with decreased influx and/or increased eflux of the drug. Aquaporin 1 (AQP1) protein has been shown to mediate the uptake of trivalent antimony. This study was aimed to find the expression level of AQP1 gene in resistant versus non-resistant clinical isolates of Leishmania major in Iranian patients.
Methods:
Clinical isolates were obtained from 16 considered patients referred to Navab Safavi Clinical Center, Isfahan, Iran from October 2014 to December 2015. After diagnosis of cutaneous leishmaniasis using microscopic observation, biopsy was performed from lesion(s) of each patient and stored inside RNAlater solution at -20C. Written informed consent was obtained from all the patients to participate in the study before recording their information and sampling based on Helsinki declaration. Each patient was treated with Glucantime and followed for three months. All sensitive and resistance isolates were considered and compared with AQP1 gene expression using real time PCR that was analyzed with delta-delta Ct.
Results:
Out of 16 clinical isolates, four patients were resistant and 12 were non-resistant. The AQP1 gene expression in resistant isolates was significantly higher than the one in response failure isolates (p = 0.001).
Interpretation & conclusion:
The significant over expression (0.5 fold) of AQP1 gene in resistant versus non- resistant isolates suggests different mechanism of drug resistance such as mutations. Mutations may change the physiological function of the Aquaporin 1 protein that might affect its expression level.
... Alternatively, inhibition of NADPHlinked pentitol dehydrogenases, which convert polyols to keto sugars for further incorporation in the pentose phosphate or glycolytic pathways, could explain the increment, as described for other organisms (Fig. 4b). However, no specific polyoldehydrogenases were described in Leishmania, although the uptake of sugar alcohols and a wide variety of other substances was previously reported (Callahan and Beverley 1992;Uzcategui et al. 2008). In the context of low carbon source in neolignan-treated promastigotes, it should be noted that glycerol-3-phosphate is also accumulated. ...
IntroductionLeishmaniasis is a parasitic neglected disease affecting millions of people worldwide. Clinical practice resorts to long and costly treatments with a therapeutic arsenal limited to highly toxic drugs, often associated to adverse side effects. Additionally, resistant strains are reported to be increasing. AimIn this work, the mechanistic action of a drug candidate (methydehydrodieugenol B), isolated from twigs of Nectandra leucantha, towards Leishmania infantum was studied by a global metabolomics approach using GC-MS and RPLC-MS platforms. MethodL. infantum promastigotes were grown in culture medium for 72 h and treated with methydehydrodieugenol B at 58.18 μg.mL-1 concentration; after 48 h treatment, enzyme activity was quenched, cells washed and frozen until analysis. For GC-MS analysis (Fiehn’s method), 1:1 methanol:water extracts were prepared and derivatized with O-methoxyamine in pyridine at room temperature for 90 min, followed by silylation with BSTFA/1% TMCS at 40 °C for 30 min. Pure methanolic extracts were also prepared and analyzed directly by RPLC-MS with a acetonitrile/water mobile phase acidulated with formic acid and gradient elution. ResultSeveral amino acids, fatty acids, carbohydrates, and glycerolipids were found as discriminant metabolites, mostly decreased in treated samples. Due to the complexity of the parasite metabolism and the great diversity of altered metabolites, a multi-target mechanism was assigned to the drug candidate, where changes in the cell energy sources and in the lipid composition of the parasite plasma membrane were prominent. Conclusion
These results contributed to elucidate the broad action of methyldehydrodieugenol B against Leishmania, paving the way in the search of novel alternative therapies.
... It has been established that other regions besides the ar/R filter and NPA motif can help in improving the fine-tuning of solute permeability. When the amino acid composition is modified, in extra-or intracellular connecting loops of aquaglyceroporins from protozoa as Dictyostelium, Plasmodium, and Leishmania, the permeability for water, glycerol or metalloids change differentially (41)(42)(43)(44). TbAQPs are 77% identical in their amino acid composition, however, TbAQP2 and 3 are phylogenetically more related (11). ...
The boron element possesses a range of different effects on living beings. It is essential to beneficial at low concentrations, but toxic at excessive concentrations. Recently, some boron-based compounds have been identified as promising molecules against Trypanosoma brucei, the causative agent of sleeping sickness. However, until now, the boron metabolism and its access route into the parasite remained elusive. The present study addressed the permeability of T. brucei aquaglyceroporins (TbAQPs) for boric acid, the main natural boron species. To this end, the three TbAQPs were expressed in Saccharomyces cerevisiae and Xenopus laevis oocytes. Our findings in both expression systems showed that all three TbAQPs are permeable for boric acid. Especially TbAQP2 is highly permeable for this compound, displaying one of the highest conductances reported for a solute in these channels. Additionally, T. brucei aquaglyceroporin activities were sensitive to pH. Taken together, these results establish that TbAQPs are channels for boric acid and are highly efficient entry pathways for boron into the parasite. Our findings stress the importance of studying the physiological functions of boron and their derivatives in T. brucei, as well as the pharmacological implications of their uptake by trypanosome aquaglyceroporins.
... Interestingly, a particular group of highly resistant isolates that clustered together were found to share genomic features associated with resistance 107 . Among these were a higher copy number for the H-locus, coding for the well-characterized ABC transporter MRPA 109 , and a homozygous two-base-pair insertion in the aquaglyceroporin 1 (AQP1) gene involved in Sb uptake and whose inactivation or downregulation is strongly correlated with resistance 71,107,[110][111][112][113][114][115][116][117] . The potential for these genomic variants in predicting treatment outcome is exciting, given the lack of molecular markers for Sb resistance, but will need to be thoroughly evaluated by using larger and geographically diversified sets of well-defined isolates. ...
Leishmania has a plastic genome, and drug pressure can select for gene copy number variation (CNV). CNVs can apply either to whole chromosomes, leading to aneuploidy, or to specific genomic regions. For the latter, the amplification of chromosomal regions occurs at the level of homologous direct or inverted repeated sequences leading to extrachromosomal circular or linear amplified DNAs. This ability of Leishmania to respond to drug pressure by CNVs has led to the development of genomic screens such as Cos-Seq, which has the potential of expediting the discovery of drug targets for novel promising drug candidates.
... Parasites can decrease the uptake of, increase the efflux of, or inactivate the drug by sequestration, among other possible mechanisms. The LmAQP1 aquaglyceroporin is the only protein known to transport antimony inside L. major (31,32), and its downregulation subsequently leads to increased drug resistance (33). Concerning efflux, members of the eukaryotic ABCC subfamily are involved in Sb III and As III resistance by exporting these metal ions outside the cells or by sequestering them within intracellular vesicles (34). ...
Treatment for Leishmaniasis, which is caused by the protozoan parasite Leishmania, currently relies on a reduced arsenal of drugs. However, the significant increase of drug therapeutic failure and the growing
resistance to first-line drugs like antimonials in some areas of Northern India and Nepal, limit the control of this parasitic
disease. Understanding the molecular mechanisms of resistance in Leishmania is now a matter of urgency to optimize drugs used and to identify novel drug targets to block or reverse resistant mechanisms.
Some members of the family of ABC (ATP-binding cassette) transporters in Leishmania have been associated with drug resistance. In this study, we have focused our interest to characterize LABCG2's involvement
in drug resistance in Leishmania. L. major parasites overexpressing the ABC protein transporter LABCG2 were generated in order to assess how LABCG2 is involved in drug
resistance. Susceptibility assays to different leishmanicidal agents were carried out. Analysis of the drug resistance profile
revealed that Leishmania parasites overexpressing LABCG2 were resistant to antimony as they demonstrated a reduced accumulation of SbIII due to an increased in drug efflux. Additionally, LABCG2 was able to transport thiols in the presence of SbIII. Biotinylation assays using parasites expressing LABCG2 fused with an N-terminal green fluorescent protein tag revealed that
LABCG2 is partially localized in the plasma membrane; this supports previous studies which suggested that LABCG2 is localized
in intracellular vesicles that fuse with the plasma membrane during exocytosis. In conclusion, Leishmania LABCG2 probably confers antimony resistance by sequestering metal-thiol conjugates within vesicles and through further exocytosis
by means of the parasite's flagellar pocket.
... The protein group can be divided into water-specific channels (orthodox AQPs), and channels that additionally facilitate transport of glycerol, urea, ammonia, and other small, uncharged molecules (aquaglyceroporins), according to specific functions of each protein. AQPs have been identified in most parasites as having roles in many physiological functions, including osmotaxis; volume regulation; and uptake of nutrients and release of toxic metabolites (Beitz, 2006;Uzcategui et al., 2008;Faghiri et al., 2010). However, little is known about AQPs in T. spiralis (TsAQP). ...
Vaccination as a preventative strategy against Trichinella spiralis infection is an ongoing effort, although no ideal vaccine candidates have been identified until now. Identification of more effective antigens that have a role in essential life stages of the parasite and that may be effective vaccine candidates is therefore of importance. In the present study, we identified a novel aquaporin gene (TsAQP) from T. spiralis, and the potential antigenicity of TsAQP was evaluated by epitope prediction. A total of 11 post-translational modification sites were predicted in the protein and fell into 4 categories: N-glycosylation; casein kinase II phosphorylation; protein kinase C phosphorylation; and N-myristoylation sites. TsAQP is a membrane intrinsic protein with high hydrophobicity; the main hydrophobic domains comprised up to 38.5% of the protein and were distributed at amino acid positions 21-43, 54-71, 83-91, 107-121, 163-174, 187-200, and 242-261. The protein consisted mainly of helices (39.58%) and loops (50%). The advanced structure of TsAQP was predicted using homology modeling, which showed that the protein was formed from 6 membrane-spanning domains connected by 5 loops. Based on these analyses, 6 potential B-cell epitopes and 4 potential T-cell epitopes were further predicted. These results suggest that TsAQP could be a promising antigen candidate for vaccination against T. spiralis.
... Another study (Uzcategui et al. 2008) examined the role of a glutamate residue, E152, located in the large extracellular hydrophilic loop positioned between transmembrane segments 3 and 4 (Fig. 2). The E152A mutation of LmAQP1 abrogates metalloid permeation but does not alter glycerol transport and thus confers a change of permeant specificity. ...
... In intracellular amastigotes, it is localized in the flagellar pocket, rudimentary flagellum, and contractile vacuoles [20]. We have shown the involvement of flexible loop C of AQP1 in determining the substrate specificity of the channel [21,22]. Additionally, we showed that AQP1 was positively regulated at the posttranslational level by a mitogen activated protein kinase 2 [17]. ...
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.
... In the absence of crystal structure, our structure-function studies have identified the critical residues of loop C for channel selectivity and function of LmAQP1. We identified that glutamate152 of loop C is responsible to discriminate between metalloids and glycerol [49]. Alanine 163 in loop C resides near the pore mouth and it is critical for the channel function, as alteration of this alanine may inactivate the channel function [50]. ...
In the class Kinetoplastida, we find an order of parasitic protozoans classified as Trypanosomatids. Three major pathogens form part of this order, Trypanosoma cruzi, Trypanosoma brucei, and Leishmania, which are responsible for disease and fatalities in millions of humans worldwide, especially in non-industrialized countries in tropical and sub-tropical regions. In order to develop new drugs and treatments, the physiology of these pathogenic protozoans has been studied in detail, specifically the significance of membrane transporters in host parasites interactions. Aquaporins and Aquaglyceroporins (AQPs) are a part of the major intrinsic proteins (MIPs) super-family. AQPs are characterized for their ability to facilitate the diffusion of water (aquaporin), glycerol (aquaglyceroporin), and other small-uncharged solutes. Furthermore, AQPs have been shown to allow the ubiquitous passage of some metalloids, such as trivalent arsenic and antimony. These trivalent metalloids are the active ingredient of a number of chemotherapeutic agents used against certain cancers and protozoan parasitic infections. Recently, the importance of the AQPs not only in osmotic adaptations but also as a factor in drug resistance of the trypanosomatid parasites has been reported. In this review, we will describe the physiological functions of aquaporins and their effect in drug response across the different trypanosomatids.
... Mechanisms contributing to drug resistance in vivo are poorly understood. However, molecules, involved in trivalent antimony or arsenite compounds transport like aquaglyceroporins (AQP1) whose down regulation provides resistance to trivalent antimony and mutation could affect specifically metal transport [93,94]. Some authors reported that terminal deletion of 67 kb to 204 kb in chromosome 31 of Leishmania, hence decreasing the copy number and expression of AQP1 led to SbIII resistance [90]. ...
Neglected diseases (NDs) are a group of diseases endemic in underdeveloped and developing countries and have been a global health problem, such as leishmaniasis, tuberculosis, cryptococcosis, leprosy among other. Drugs used in the clinic are toxic, they do not always result in a cure, and many parasites have shown resistance to them. In the last years, the incidence and prevalence rates of some Neglected Diseases have decreased in the world, opposite to the multi-drugresistance (MDT) levels observed. Drug resistance in leprosy and tuberculosis becomes even more important because they’re very limited alternative drugs to MDT. Molecular studies on the mechanism of action of these drugs have elucidated the genetic basis of drug resistance in Mycobacterium tuberculosis and Mycobacterium leprae, respectively. C. neoformans has been shown to present different susceptibility profiles to antifungal drugs according to in vitro studies, although little reports of resistant cases have been described. C. neoformans resistance can be seen to azoles, especially fluconazole, to amphotericin B (AmB) and to 5-flucytosine (5FC). This chapter will summarize the main mechanisms of resistance of some neglected diseases such as leishmaniasis, leprosy, cryptococcosis and other.
... Regulation is attained by LmjMPK2 phosphorylating LmjAQP1 at resides in the small cytosolic loop D between transmembrane domains 4 and 5 of LmjAQP1 (cf. Uzcategui et al., 2008). Consequent to overexpression and phosphorylation of wild-type LmjAQP1, a higher intracellular accumulation of Sb(III) ( Fig. 2A and B) and hypersensitivity to Sb(III) was noted (Table 1). ...
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.
... In fact, the replacement of alanine at position 163 by serine or threonine induced the decrease of the entry of the active form of antimony [70]. Other mutations in the sequence of this protein such as an alteration at Glu152 or Arg230 were associated with reducing SbIII entry [71]. These mutations were not found in resistant Indian clinical isolates with an underexpression of AQP1 [72]. ...
Leishmaniases are parasitic diseases that spread in many countries with a prevalence of 12 million cases. There are few available treatments and antimonials are still of major importance in the therapeutic strategies used in most endemic regions. However, resistance toward these compounds has recently emerged in areas where the replacement of these drugs is mainly limited by the cost of alternative molecules. In this paper, we reviewed the studies carried out on antimonial resistance in Leishmania. Several common limitations of these works are presented before prevalent approaches to evidence antimonial resistance are related. Afterwards, phenotypic determination of resistance is described, then confronted to clinical outcome. Finally, we detail molecular mechanisms and targets involved in resistance and already identified in vitro within selected mutant strains or in clinical isolates.
... Gene amplification has been also described in Leishmania strains selected for resistance to methotrexate [11]. Other molecules are involved in trivalent antimony or arsenite compounds transport like aquaglyceroporins whose down regulation provides resistance to trivalent antimony and mutation could affect specifically metal transport [12]. However, all these findings derived from studies of strains whose resistance was induced in vitro and data about natural conditions are lacking. ...
Experimental studies about Leishmania resistance to metal and antifolates have pointed out that gene amplification is one of the main mechanisms of drug detoxification. Amplified genes code for adenosine triphosphate-dependent transporters (multidrug resistance and P-glycoproteins P), enzymes involved in trypanothione pathway, particularly gamma glutamyl cysteine synthase, and others involved in folates metabolism, such as dihydrofolate reductase and pterine reductase. The aim of this study was to detect and quantify the amplification of these genes in clinical strains of visceral leishmaniasis agents: Leishmania infantum, L. donovani, and L. archibaldi. Relative quantification experiments by means of real-time polymerase chain reaction showed that multidrug resistance gene amplification is the more frequent event. For P-glycoproteins P and dihydrofolate reductase genes, level of amplification was comparable to the level observed after in vitro selection of resistant clones. Gene amplification is therefore a common phenomenon in wild strains concurring to Leishmania genomic plasticity. This finding, which corroborates results of experimental studies, supports a better understanding of metal resistance selection and spreading in endemic areas.
Cholesterol is an essential component of the plasma membrane in mammalian cells. It plays critical roles in numerous cellular functions, including cell signaling, as well as in cell viability, growth, and proliferation. A variety of methods for altering the levels of cholesterol at the cell, tissue, and organism levels have been developed to identify, define, and characterize such roles. In this chapter, we describe the key in vitro and in vivo approaches used for this purpose and the challenges involved in their application. The approaches under consideration include the application of liposomes to enrich Xenopus oocytes, the use of cyclodextrins to either enrich or deplete oocytes, cells, and tissues, the utilization of lipoproteins to alter the levels of cholesterol in cells and tissues, and the administration of specific diets and therapies that affect cholesterol levels in laboratory animals. The chapter concludes with examples demonstrating the results of applying a variety of these approaches in ion channel research.
PurposeLeishmaniasis comprises various clinical forms mainly including cutaneous, muco-cutaneous, and visceral leishmaniasis; caused by Leishmania species. Antimoniate is the first-line treatment but some cases showed no response to treatment in the worldwide. In this study, mitogen-activated protein kinase (MAPK) and aquaglyceroporin 1 (AQP1) gene expressions were assessed in treatment failure clinical isolates of Leishmania major. Also, molecular and phylogenic analyses of the mentioned isolates were performed.Methods
Samples were obtained from the patients with suspicious CL referred to the laboratory of Diagnosis Center, Gorgan Province, Iran, from October 2016 to December 2019. Detection and identification of the parasite was performed. The genes expressions of MAPK1 and AQP1 were done using SYBR Green real-time PCR. The AQP1 gene from the isolates with treatment failure was sequenced and analyzed using BLAST and multiple alignments. The phylogenic analysis was done using MEGA7. The statistical analysis was done using SPSS 16.0 by non-parametric Mann–Whitney U test.ResultsAll clinical isolates were detected L. major. The mean AQP1 and MAPK1 gene expressions in treatment failure isolates were 58.71 and 6.139 fold less than the ones in treatment response isolates, respectively. Based on the AQP1 gene sequence, a nucleotide change of aspartic acid with asparagine at the site 234 was observed. Phylogenic tree analysis showed three groups with the minimum dissimilarity of 0.008 between TF isolates with the standard L. major strains.Conclusion
We showed that MAPK1 and AQP1 may have critical roles in response to antimoniate in clinical isolates L. major in this study.
Aquaporins are membrane proteins responsible for permeating water, ions, dissolved gases, and other small molecular weight compounds through the protective cell membranes of living organisms. These proteins have been gaining increased importance as targets for treating a variety of parasitic diseases, since they control key physiological processes in the life cycle of parasitic protozoans, such as the uptake of nutrients, release of metabolites, and alleviation of osmotic stress. In this work, we use homology modeling to build three-dimensional structures for the four main aquaporins encoded and expressed by Leishmania major, a protozoan that causes leishmaniasis and affects millions of people worldwide. Physico-chemical properties of the proposed models for LmAQP1, LmAQPα, LmAQPβ, and LmAQPγ are then investigated using molecular dynamics simulations and the reference interaction site model (RISM) molecular theory of solvation. Pore characteristics, water permeation, and potential of mean force across the AQP channels for water, methanol, urea, ammonia, and carbon dioxide are examined and compared with results obtained for a protozoan (Plasmodium falciparum) aquaporin for which a crystal structure is available.
Trichinella spiralis, the causative agent of trichinellosis, is able to infect a wide range of carnivores and omnivores including human beings. In the past 30 years, a mass of vaccination efforts has been performed to control T. spiralis infection with the purpose of reduction in worm fecundity or decrease in muscle larval and adult burdens. Here, we summarize the development of veterinary vaccines against T. spiralis infection. During recent years, increasing numbers of new vaccine candidates have been developed on the protective immunity against T. spiralis infection in murine model. The vaccine candidates were not only selected from excretory–secretory (ES) antigens, but also from the recombinant functional proteins, such as proteases and some other antigens participated in T. spiralis intracellular processes. However, immunization with a single antigen generally revealed lower protective effects against T. spiralis infection in mice compared to that with the inactivated whole worms or crude extraction and ES productions. Future study of T. spiralis vaccines should focus on evaluation of the protective efficacy of antigens and/or ligands delivered by nanoparticles that could elicit Th2‐type immune response on experimental pigs.
The vestibule loop regions of aquaglyceroporins are involved in accumulation of glycerol inside the channel pore. Even though most loop regions do not show high sequence similarity among aquaglyceroporins, loop E is highly conserved in aquaglyceroporins, but not in members of the homologous aquaporins. Specifically, a tryptophan residue is extremely conserved within this loop. We have investigated the role of this residue (Trp219) that deeply protrudes into the protein and potentially interacts with adjacent loops, using the E. coli aqualgyeroporin GlpF as a model. Replacement of Trp219 affects the activity of GlpF and impairs the stability of the tetrameric protein. Furthermore, we have identified an amino acid cluster involving Trp219 that stabilizes the GlpF tetramer. Based on our results we propose that Trp219 is key for formation of a defined vestibule structure, which is crucial for glycerol accumulation as well as for the stability of the active GlpF tetramer. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.
Leishmania parasites rely heavily upon membrane transport proteins to deliver essential nutrients from their hosts to the interior of the parasite. Some of these transporters also serve as routes for uptake of drugs used for treatment of leishmaniasis or experimental drugs with potential for development of novel anti-leishmanial therapies. Hence, mutations within the coding regions of such permeases or alterations in the expression of the carrier proteins can confer drug resistance upon the parasites. This chapter reviews the current level of knowledge regarding several classes of membrane transporters known to play roles in uptake or sensitivity to drugs. The increasing knowledge of the “permeome,” provided by complete genome sequences of several Leishmania species, has advanced considerably our knowledge of how nutrients and drugs or other cytotoxic compounds enter these pathogenic protozoa.
IntroductionMetalloid Uptake SystemsMetalloid Efflux SystemsSummary and Conclusions
AcknowledgementsReferences
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. © 2013 S. Karger AG, Basel.
Aquaporins (AQPs) are widely distributed in all kingdoms of life and act as facilitators in the transport of water and other small solutes through cell membranes. Since the plasmodial and human AQPs are different in their primary and secondary structure, an intervention targeting plasmodial AQP without affecting human AQPs is discussed to identify an attractive novel target against malaria. Therefore, it is crucial to understand the action mechanisms of these plasmodial AQPs. To explore the progression of the plasmodial real AQPs in vivo at work, a molecular dynamic simulation system was successfully developed for a PfAQP tetramer in silico. The results showed that the transporting work was not synchronous in the four channels at the same time, and that it was different at different times in the same channel. The hole sizes varied in different channels with time. The structure analysis showed that both hydrophobic and hydrophilic residues composed the inner surface of the channels, and the asparagines Asn-193 and Asn-70 assembled into two motifs of NLA and NPS in the center of the channel in place of the signature motifs of NPA in other AQPs. In brief, we successfully developed an equilibrated PfAQP-lipid system by molecular dynamics simulations, and investigated the structure of the PfAQP channel, which should aid our understanding of the AQP structure and its functional implications.
Aquaporins are important channel proteins that are responsible for the balance of cellular osmolarity and nutrient transport in vertebrates. Recently, new functions of these ancient channels have been found in the conduction of metalloid arsenic (As). Chronic As exposure through contaminated water and food sources is associated with multiple human diseases and endangers millions of people's health worldwide. Therefore, identification of the As transport pathways is necessary to elucidate the mechanisms of As carcinogenesis. Arsenic detoxification systems have been studied in multiple vertebrates such as mammalian mouse, rat, humans and nonmammalian vertebrates. Multiple transporters and enzymes have been shown to be involved in As translocation and cellular transformation. In these vertebrates, members ofaquaglyceroporins, which include AQP7 in kidney and AQP9 in liver, catalyze uptake of inorganic trivalent arsenite [As(III)]. AQP9, the major liver aquaglyceroporin, conducts both inorganic As(III) and organic monomethylarsonous acid [MMA(III)], an intermediate that is generated during the cellular methylation. As a channel that facilitates a downhill movement of substances dependent on the concentration gradient, AQP9 may play an important role in the simultaneous influx of inorganic As(III) from blood to liver and efflux of As metabolite MMA(III) from liver to blood. In this chapter, we will discuss the function ofaquaglyceroporins ofvertebrates in uptake and detoxification of the metalloid As.
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.
Clinical resistance to pentavalent antimonials results from an interplay between uptake, efflux and sequestration in Leishmania. Aquaglyceroporins (AQPs) have been shown to facilitate uptake of trivalent metalloids. Down-regulation of AQP1 in Leishmania results in resistance to trivalent antimony, whereas overexpression of AQP1 in drug-resistant parasites can reverse the resistance. The present work investigates the role of AQP1 in monitoring antimonial resistance in Indian leishmaniasis.
Susceptibility to trivalent antimony as determined in vitro with intracellular amastigotes from both visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients correlated well with the clinical response. Higher accumulation of trivalent antimony (SbIII) was observed in all susceptible isolates compared with resistant isolates. Reduced accumulation of SbIII correlated, with a few exceptions, with down-regulation of AQP1 RNA as determined by real-time PCR. Cloning and sequencing of the AQP1 gene from both VL and PKDL isolates showed sequence variation in four of the clinical isolates. None of the isolates had an alteration of Glu152 and Arg230, which have been previously shown to affect metalloid transport. Transfection of the AQP1 gene in a sodium antimony gluconate-resistant field isolate conferred susceptibility to the resistant isolate.
Our studies indicate genetic variation in VL and PKDL isolates. Down-regulation of AQP1 correlates well with clinical drug resistance in a majority of Indian VL and PKDL isolates. AQP1 gene expression at both the genetic and transcriptional level showed positive correlation with SbIII accumulation, with some exceptions.
Protozoan parasites are a major threat to human health with millions of fatalities worldwide, especially in nonindustrialized countries. Currently, there is no cure for many of these parasitic diseases. Consequently, there is an imperative to find treatment targets and develop novel drugs based on the proteins encoded in the genomes of these parasites. Aquaporins, members of membrane proteins discovered and characterized within the past 20 years, are the mechanism through which water is transported through living membranes. The presence of aquaporins explains disease etiology related to water physiology and presents new pharmacogenomic targets. In this article, we review the literature on aquaporins found in Apicomplexan, Kinetoplastida and Microsporidia parasites as potential drug targets. Furthermore, by analyzing protein motion dynamics, we identify impediments that need to be surmounted for developing effective drugs targeting the aquaglyceroporin of Plasmodium falciparum, the causative agent of the most fatal form of human malaria.
The protozoan flagellate Leishmania donovani has an active myo-inositol/proton symporter (MIT), which is driven by a proton gradient across the parasite membrane. We have used site-directed
mutagenesis in combination with functional expression of transporter mutants in Xenopusoocytes and overexpression in Leishmania transfectants to investigate the significance of acidic transmembrane residues for proton relay and inositol transport. MIT
has only three charged amino acids within predicted transmembrane domains. Two of these residues, Asp19 (TM1) and Glu121 (TM4), appeared to be critical for transport function of MIT, with a reduction of inositol transport to about 2% of wild-type
activity when mutated to the uncharged amides D19N or E121Q and 20% (D19E) or 4% (E121D) of wild-type activity for the conservative
mutations that retained the charge. Immunofluorescence microscopy of oocyte cryosections showed that MIT mutants were expressed
on the oocyte surface at a similar level as MIT wild type, confirming that these mutations affect transport function and do
not prevent trafficking of the transporter to the plasma membrane. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone
and dinitrophenol inhibited inositol transport by 50–70% in the wild-type as well as in E121Q, despite its reduced transport
activity. The mutant D19N, however, was stimulated about 4-fold by either protonophore and 2-fold by cyanide or increase of
pH 7.5 to 8.5 but inhibited at pH 6.5. The conservative mutant D19E, in contrast, showed an inhibition profile similar to
MIT wild type. We conclude that Asp19 and Glu121 are critical for myo-inositol transport, while the negatively charged carboxylate at Asp19 may be important for proton coupling of MIT.
The malaria parasite Plasmodium falciparum faces drastic osmotic changes during kidney passages and is engaged in the massive biosynthesis of glycerolipids during its
development in the blood-stage. We identified a single aquaglyceroporin (PfAQP) in the nearly finished genome of P. falciparum with highest similarity to the Escherichia coli glycerol facilitator (50.4%), but both canonical Asn-Pro-Ala (NPA) motifs in the pore region are changed to Asn-Leu-Ala (NLA)
and Asn-Pro-Ser (NPS), respectively. Expression in Xenopusoocytes renders them highly permeable for both water and glycerol. Sugar alcohols up to five carbons and urea pass the pore.
Mutation analyses of the NLA/NPS motifs showed their structural importance, but the symmetrical pore properties were maintained.
PfAQP is expressed in blood-stage parasites throughout the development from rings via trophozoites to schizonts and is localized
to the parasite but not to the erythrocyte cytoplasm or membrane. Its unique bi-functionality indicates functions in the protection
from osmotic stress and efficiently provides access to the serum glycerol pool for the use in ATP generation and primarily
in the phospholipid synthesis.
Aquaglyceroporins form the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes. AQP9 has unusually broad solute permeability and is expressed in hepatocyte plasma membranes. Proteoliposomes reconstituted with expressed, purified rat AQP9 protein were compared with simple liposomes for solute permeability. At pH 7.5, AQP9 proteoliposomes exhibited Hg(2+)-inhibitable glycerol and urea permeabilities that were increased 63-fold and 90-fold over background. beta-Hydroxybutyrate permeability was not increased above background, and osmotic water permeability was only minimally elevated. During starvation, the liver takes up glycerol for gluconeogenesis. Expression of AQP9 in liver was induced up to 20-fold in rats fasted for 24-96 h, and the AQP9 level gradually declined after refeeding. No changes in liver AQP9 levels were observed in rats fed ketogenic diets or high-protein diets, but AQP9 levels were elevated in livers of rats made diabetic by streptozotocin injection. When blood glucose levels of the diabetic rats were restored to normal by insulin treatments, the AQP9 levels returned to baseline. Confocal immunofluorescence revealed AQP9 immunostaining on the sinusoidal surfaces of hepatocyte plates throughout the livers of control rats. Denser immunostaining was observed in the same distribution in livers of fasted and streptozotocin-treated rats. We conclude that AQP9 serves as membrane channel in hepatocytes for glycerol and urea at physiological pH, but not for beta-hydroxybutyrate. In addition, levels of AQP9 expression fluctuate depending on the nutritional status of the subject and the circulating insulin levels.
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.
Arsenic, which is ubiquitous in the environment and comes from both geochemical and anthropogenic sources, has become a worldwide
public health problem. Every organism studied has intrinsic or acquired mechanisms for arsenic detoxification. In Saccharomyces cerevisiae arsenate is detoxified by Acr2p, an arsenate reductase. Acr2p is not a phosphatase but is a homologue of CDC25 phosphatases.
It has the HCX5R phosphatase motif but not the glycine-rich phosphate binding motif (GXGXXG) that is found in protein-tyrosine phosphatases. Here we show that creation of a phosphate binding motif through the introduction
of glycines at positions 79, 81, and 84 in Acr2p resulted in a gain of phosphotyrosine phosphatase activity and a loss of
arsenate reductase activity. Arsenate likely achieved geochemical abundance only after the atmosphere became oxidizing,
creating pressure for the evolution of an arsenate reductase from a protein-tyrosine phosphatase. The ease by which an arsenate
reductase can be converted into a protein-tyrosine phosphatase supports this hypothesis.
Silicon is beneficial to plant growth and helps plants to overcome abiotic and biotic stresses by preventing lodging (falling over) and increasing resistance to pests and diseases, as well as other stresses. Silicon is essential for high and sustainable production of rice, but the molecular mechanism responsible for the uptake of silicon is unknown. Here we describe the Low silicon rice 1 (Lsi1) gene, which controls silicon accumulation in rice, a typical silicon-accumulating plant. This gene belongs to the aquaporin family and is constitutively expressed in the roots. Lsi1 is localized on the plasma membrane of the distal side of both exodermis and endodermis cells, where casparian strips are located. Suppression of Lsi1 expression resulted in reduced silicon uptake. Furthermore, expression of Lsi1 in Xenopus oocytes showed transport activity for silicon only. The identification of a silicon transporter provides both an insight into the silicon uptake system in plants, and a new strategy for producing crops with high resistance to multiple stresses by genetic modification of the root's silicon uptake capacity.
Boron (B) is essential in plants but often present at low concentrations in the environment. To investigate how plants survive under conditions of B limitation, we conducted a transcriptome analysis and identified NIP5;1, a member of the major intrinsic protein family, as a gene upregulated in B-deficient roots of Arabidopsis thaliana. Promoter-beta-glucuronidase fusions indicated that NIP5;1 is strongly upregulated in the root elongation zone and the root hair zone under B limitation, and green fluorescent protein-tagged NIP5;1 proteins localized to the plasma membrane. Expression in Xenopus laevis oocytes demonstrated that NIP5;1 facilitated the transport of boric acid in addition to water. Importantly, two T-DNA insertion lines of NIP5;1 displayed lower boric acid uptake into roots, lower biomass production, and increased sensitivity of root and shoot development to B deficiency. These results identify NIP5;1 as a major plasma membrane boric acid channel crucial for the B uptake required for plant growth and development under B limitation.
The 2.05-A resolution structure of the aquaglyceroporin from the malarial parasite Plasmodium falciparum (PfAQP), a protein important in the parasite's life cycle, has been solved. The structure provides key evidence for the basis of water versus glycerol selectivity in aquaporin family members. Unlike its closest homolog of known structure, GlpF, the channel conducts both glycerol and water at high rates, framing the question of what determines high water conductance in aquaporin channels. The universally conserved arginine in the selectivity filter is constrained by only two hydrogen bonds in GlpF, whereas there are three in all water-selective aquaporins and in PfAQP. The decreased cost of dehydrating the triply-satisfied arginine cation may provide the basis for high water conductance. The two Asn-Pro-Ala (NPA) regions of PfAQP, which bear rare substitutions to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), participate in preserving the orientation of the selectivity filter asparagines in the center of the channel.
Arsenic is a toxic and highly abundant metalloid that endangers human health through drinking water and the food chain. The most common forms of arsenic in the environment are arsenate (As(V)) and arsenite (As(III)). As(V) is a non-functional phosphate analog that enters the food chain via plant phosphate transporters. Inside cells, As(V) becomes reduced to As(III) for subsequent extrusion or compartmentation. Although much is known about As(III) transport and handling in microbes and mammals, the transport systems for As(III) have not yet been characterized in plants.
Here we show that the Nodulin26-like Intrinsic Proteins (NIPs) AtNIP5;1 and AtNIP6;1 from Arabidopsis thaliana, OsNIP2;1 and OsNIP3;2 from Oryza sativa, and LjNIP5;1 and LjNIP6;1 from Lotus japonicus are bi-directional As(III) channels. Expression of these NIPs sensitized yeast cells to As(III) and antimonite (Sb(III)), and direct transport assays confirmed their ability to facilitate As(III) transport across cell membranes. On medium containing As(V), expression of the same NIPs improved yeast growth, probably due to increased As(III) efflux. Our data furthermore provide evidence that NIPs can discriminate between highly similar substrates and that they may have differential preferences in the direction of transport. A subgroup of As(III) permeable channels that group together in a phylogenetic tree required N-terminal truncation for functional expression in yeast.
This is the first molecular identification of plant As(III) transport systems and we propose that metalloid transport through NIPs is a conserved and ancient feature. Our observations are potentially of great importance for improved remediation and tolerance of plants, and may provide a key to the development of low arsenic crops for food production.
Arsenic poisoning affects millions of people worldwide. Human arsenic intake from rice consumption can be substantial because rice is particularly efficient in assimilating arsenic from paddy soils, although the mechanism has not been elucidated. Here we report that two different types of transporters mediate transport of arsenite, the predominant form of arsenic in paddy soil, from the external medium to the xylem. Transporters belonging to the NIP subfamily of aquaporins in rice are permeable to arsenite but not to arsenate. Mutation in OsNIP2;1 (Lsi1, a silicon influx transporter) significantly decreases arsenite uptake. Furthermore, in the rice mutants defective in the silicon efflux transporter Lsi2, arsenite transport to the xylem and accumulation in shoots and grain decreased greatly. Mutation in Lsi2 had a much greater impact on arsenic accumulation in shoots and grain in field-grown rice than Lsi1. Arsenite transport in rice roots therefore shares the same highly efficient pathway as silicon, which explains why rice is efficient in arsenic accumulation. Our results provide insight into the uptake mechanism of arsenite in rice and strategies for reducing arsenic accumulation in grain for enhanced food safety.
In several Leishmania spp., resistance to methotrexate and other drugs is often associated with amplification of the chromosomal H region in the form of extrachromosomal H circles. We report here that the H circle of Leishmania tarentolae contains an 867 bp open reading frame, ltdh, which mediates high levels of resistance to methotrexate and other antifolates, after transfection. The predicted amino acid sequence of the ltdh gene product has significant similarities to a family of short-chain dehydrogenases, enzymes that are involved in several oxido-reduction reactions in a wide range of organisms. To resist antifolates, Leishmania amplifies the ltdh gene as part of the H circle. We propose that LTDH might be involved in an alternative pathway for the synthesis of reduced folates and that ltdh overproduction represents a novel mechanism for resistance to antifolates. Our results support the hypothesis that the H region of the Leishmania genome contains several drug resistance genes and that preferential amplification of this region has evolved as a defense mechanism against cytotoxic drugs.
Acquired resistance to methotrexate in Leishmania species is often associated with the amplification of H circles, 68 kb duplex DNA circles containing a 30 kb inverted repeat. We report here that the H circle of Leishmania tarentolae contains an open reading frame, ltpgpA, that has the attributes of P-glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). Although amplification of H circles is associated with proportionally increased levels of a 5.5 kb transcript of the ltpgpA gene, such methotrexate resistant mutants are not cross-resistant to any of the drugs extruded by mammalian multi-drug resistant cells. In Leishmania, ltpgpA is part of a gene family containing at least two other members. Sequences homologous to one of the nucleotide binding sites of ltpgpA are conserved in other kinetoplastida.
Unlabelled:
T(E)Xtopo is a LAT(E)X2epsilon macro package for plotting topology data directly from PHD predictions or SwissProt database files in publication-ready quality. The plot can be shaded automatically to emphasize conserved residues or functional properties of the residue sidechains. The addition of rich decorations, such as labels, annotations and legends, is easily accomplished.
Availability:
The T(E)Xtopo macro package and a full on-line documentation are freely available at http://homepages.uni-tuebingen.de/beitz/
Contact:
eric.beitz@uni-tuebingen.de
A regulatory volume decrease (RVD) in response to hyposmotic stress has been characterized in different life-cycle stages of Trypanosoma cruzi. Hyposmotic stress initially caused swelling, but this was rapidly reversed by a compensatory volume reversal that was essentially complete by 5 min. Volume recovery was associated with an amino acid efflux that accounted for approximately 50% of the regulatory volume decrease in all three life-cycle stages. The amino acid efflux was selective for neutral and anionic amino acids, but excluded cationic amino acids. Acidocalcisomes contained an amino acid pool over four times more concentrated than whole-cell levels, but about 90% of this was composed of Arg and Lys, so involvement of this pool in amino acid efflux was ruled out. Hyposmotic stress induced a rise in intracellular calcium that was dependent on influx of calcium across the plasma membrane, since chelation of extracellular calcium abolished the response. Influx of calcium was confirmed by demonstration of manganese-mediated quenching of intracellular fura-2 fluorescence and partial inhibition of the rise in calcium by calcium channel blockers. Manipulation of intra- and extracellular calcium levels had minor effects on the initial rate of amino acid efflux and no effect on the rate of volume recovery.
During the insect phase of the parasite lifecycle, Leishmania promastigotes move from the midgut to the anterior regions of the alimentary tract of their sandfly vector. Chemotaxis of Leishmania promastigotes towards sugars has been reported, and the putative presence of sugar gradient in the insect foregut has been suggested to play a role in promastigote development in the insect. We have further investigated the potential of Leishmania mexicana promastigotes to respond to chemical stimulii. We find that promastigotes move towards concentrations of all substances tested and that this taxis requires the presence of an osmotic gradient. Our results indicate that behaviour that has previously been interpreted as chemotaxis is better understood as osmotaxis. The implications of this observation are discussed in the context of promastigote development.
Glycoconjugates have been shown to play important roles in Leishmania development. However, the ability to study these molecules and other processes would benefit greatly from improved methods for genetic manipulation and analysis of the amastigote stage. This is especially challenging for L. donovani, the agent of the most severe form of leishmaniasis, which can rapidly lose virulence during in vitro culture. Here we report on a clonal subline of an L. donovani 1S2D (LdBob or LdB), which differentiates readily from promastigotes to amastigotes in axenic culture, and maintains this ability during extended parasite cultivation in vitro. This derivative can be plated and transfected efficiently while grown as promastigotes or amastigotes. Importantly, LdB maintains the ability to differentiate while undergoing genetic alterations required for creation of gene knockouts and complemented lines. Like virulent L. donovani, LdB exhibits down-regulation of lipophosphoglycan (LPG) synthesis and up-regulation of A2 protein synthesis in amastigotes. We showed that knockouts of LPG2, encoding a Golgi GDP-mannose transporter, eliminated phosphoglycan synthesis in LdB axenic amastigotes. These and other data suggest that LdB axenic amastigotes will be generally useful as a differentiation model in studies of gene expression, virulence, glycoconjugate function and drug susceptibility in L. donovani.
Long-term exposure to arsenic in drinking water has been linked to cancer of the bladder, lungs, skin, kidney, nasal passages, liver, and prostate in humans. It is therefore important to understand the structural aspects of arsenic in water, as hydrated arsenic is most likely the initial form of the metalloid absorbed by cells. We present a detailed experimental and theoretical characterization of the coordination environment of hydrated arsenite. XANES analysis confirms As(III) is a stable redox form of the metalloid in solution. EXAFS analysis indicate, at neutral pH, arsenite has a nearest-neighbor coordination geometry of approximately 3 As-O bonds at an average bond length of 1.77 A, while at basic pH the nearest-neighbor coordination geometry shifts to a single short As-O bond at 1.69 A and two longer As-O bonds at 1.82 A. Long-range ligand scattering is present in all EXAFS samples; however, these data could not be fit with any degree of certainty. There is no XAS detectable interaction between As and antimony, suggesting they are not imported into cells as a multinuclear complex. XAS results were compared to a structural database of arsenite compounds to confirm that a 3 coordinate As-O complex for hydrated arsenite is the predominate species in solution. Finally, quantum chemical studies indicate arsenite in solution is solvated by 3 water molecules. These results indicate As(OH)3 as the most stable structure existing in solution at neutral pH; thus, ionic As transport does not appear to be involved in the cellular uptake process.
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.
The selectivity of aquaporins for water and solutes is determined by pore diameter. Paradoxically, the wider pores of glycerol facilitators restrict water passage by an unknown mechanism. Earlier we characterized an aquaglyceroporin from Plasmodium falciparum with high permeability for both glycerol and water. We use point mutations to demonstrate that amino acids directly lining the pore are not responsible for the excellent water permeability of the Plasmodium aquaglyceroporin but affect permeability of pentitols. Within a conserved WET triad in the extracellular C-loop we identified a Plasmodium aquaglyceroporin-specific glutamate (E125) located in proximity to a conserved arginine (R196) at the pore mouth. Mutation of E125 to serine largely abolished water permeability. Concomitantly, the activation energy for water permeation was increased by 4 kcal/mol. Mutation of the adjacent tryptophan to cysteine led to irreversible inhibition of water passage by Hg(2+). This unequivocally proves the proximity of the couple W124/E125 close to the pore mouth. We conclude that in the Plasmodium aquaglyceroporin the electrostatic environment at the extracellular pore entry regulates water permeability.
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.
Arsenic toxicity has become a global concern owing to the ever-increasing contamination of water, soil and crops in many regions of the world. To limit the detrimental impact of arsenic compounds, efficient strategies such as phytoremediation are required. Suitable plants include arsenic hyperaccumulating ferns and aquatic plants that are capable of completing their life cycle in the presence of high levels of arsenic through the concerted action of arsenate reduction to arsenite, arsenite complexation, and vacuolar compartmentalization of complexed or inorganic arsenic. Tolerance can also be conferred by lowering arsenic uptake by suppression of phosphate transport activity, a major pathway for arsenate entry. In many unicellular organisms, arsenic tolerance is based on the active removal of cytosolic arsenite while limiting the uptake of arsenate. Recent molecular studies have revealed many of the gene products involved in these processes, providing the tools to improve crop species and to optimize phytoremediation; however, so far only single genes have been manipulated, which has limited progress. We will discuss recent advances and their potential applications, particularly in the context of multigenic engineering approaches.
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.
Recent experimental results suggest that As(III) and Sb(III) transport in prokaryotes and eukaryotes might be facilitated by aquaglyceroporins. GlpF, the glycerol facilitator in Escherichia coli was the first to be identified as a trivalent metalloid transporter. Quantum calculations have been performed to study the possible existence of common structural properties between As(OH) 3 and Sb(OH) 3 and glycerol. Because the mechanism of substrate migration is primarily related to the successive formation of hydrogen bonds between the substrate and the hydrophilic part of the channel wall, this study has focused on the structural, thermodynamic, and electrostatic comparison of the main As(III) and Sb(III) compounds present in aqueous solution at physiological pH values, As(OH) 3 and Sb(OH) 3, with the glycerol conformation closest to the structures of these As- and Sb-containing compounds. This particular glycerol conformation has then been compared to three known experimental conformations of glycerol present in the protein channel. Besides their stoichiometry and electroneutral condition, As(OH) 3 and Sb(OH) 3 show very strong similarities to both each other and the studied conformation of the glycerol molecule: Namely, they show a similar charge distribution and a slightly smaller volume than glycerol. Their smaller size can be an additional advantage for the transit through the narrowest region of the GlpF channel. However, the metalloid hydroxyl groups lack the flexibility of glycerol, which probably helps this molecule to adapt its conformation to the topology of the GlpF channel.
We studied the effect of loss of function in the NIP subfamily II in Arabidopsis thaliana to assess their potential role(s) in arsenite (AsIII) uptake. Loss of function in AtNIP7;1 led to increased plant tolerance to AsIII and reduced total As in planta. AtNIP7;1 expression in various yeast backgrounds increased AsIII sensitivity. In the acr3Delta yeast genotype, AtNIP7;1 caused a moderate increase in AsV tolerance. Short-term As uptake in fsp1Delta expressing AtNIP7;1 was significantly larger than that in the empty vector control. The data suggest that AtNIP7;1 can mediate AsIII transport and contributes to AsIII uptake in plants.
Each mutation was confirmed by sequencing the References Beitz
- ′-G E152q
- ′-Ctt Gga Cgc Cat Cgt Ttg Att Cag Aag Caa C-3′ T E Ttg Ctt Ctg Aat Caa Acg Atg Gcg Tcc Aag-3′
- Xtopo
E152Q, 5′-G TTG CTT CTG AAT CAA ACG ATG GCG TCC AAG-3′ (sense) and 5′-CTT GGA CGC CAT CGT TTG ATT CAG AAG CAA C-3′ (antisense). Each mutation was confirmed by sequencing the References Beitz, E. (2000) T E Xtopo: shaded membrane protein topology plots in L A T E X2e. Bioinformatics 16: 1050–1051.
Molecular Cloning: a Laboratory Manual. Cold Spring Harbor
- J Sambrook
- D Russell
Sambrook, J., and Russell, D. (2001) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
The mutagenic oligonucleotides used for both strands and the respective changes introduced (underlined) are as follows
- E143a 145a
- ′-G Ctc Ttc Aaa Tcg Cac Ttt Gat Gca Gcc Gca Aag Agg Ttg Ctt Ctg-3′
- ′-Cag Aag Caa Cct Ctt Tgc Ggc Tgc Atc Aaa Gtg Cga Ttt Gaa Gag
2003). The mutagenic oligonucleotides used for both strands and the respective changes introduced (underlined) are as follows: E143A/145A, 5′-G CTC TTC AAA TCG CAC TTT GAT GCA GCC GCA AAG AGG TTG CTT CTG-3′ (sense) and 5′-CAG AAG CAA CCT CTT TGC GGC TGC ATC AAA GTG CGA TTT GAA GAG C-3′ (antisense);