Drug Uptake and Modulation of Drug Resistance in Leishmania by an Aquaglyceroporin

Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field. 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. Leishmaniasis is a parasitic disease caused by the protozoan parasite Leishmania sp. Leishmaniasis is a spectrum of diseases in which each disease is distinctly manifested, and all have potentially devastating consequences: disfigurement, damage to internal organs, and death (1Choi C.M. Lerner E.A. Am. J. Clin. Dermatol. 2002; 3: 91-105Google Scholar). In the last two decades, visceral leishmaniasis especially has been widely recognized as an opportunistic infection in immunocompromised patients, particularly those who are infected with human immunodeficiency virus (1Choi C.M. Lerner E.A. Am. J. Clin. Dermatol. 2002; 3: 91-105Google Scholar, 2Silva E.S. Pacheco R.S. Gontijo C.M. Carvalho I.R. Brazil R.P. Rev. Inst. Med. Trop. Sao Paulo. 2002; 44: 145-149Google Scholar). The first line choice of treatment against all forms of leishmaniasis uses drugs containing pentavalent antimony such as sodium stibogluconate (Pentostam) and meglumine antimonate (Glucantime). Clinical resistance to this class of drugs, however, has been reported from many parts of the world (3Guerin P.J. Olliaro P. Nosten F. Druilhe P. Laxminarayan R. Binka F. Kilama W.L. Ford N. White N.J. Lancet Infect. Dis. 2002; 2: 564-573Google Scholar). More than 50% of the cases of visceral leishmaniasis in Northeast India are resistant to pentavalent antimonials (4Sundar S. More D.K. Singh M.K. Singh V.P. Sharma S. Makharia A. Kumar P.C. Murray H.W. Clin. Infect. Dis. 2000; 31: 1104-1107Google Scholar). Resistance to pentavalent antimonials in Leishmania is a priority of the World Health Organization (www.who.int/infectious-disease-report/2000). Leishmania resistant to trivalent antimony has also been reported in vitro, which has provided an excellent model for the study of resistance mechanisms (5Ouellette M. Legare D. Haimeur A. Grondin K. Roy G. Brochu C. Papadopoulou B. Drug Resist. Updat. 1998; 1: 43-48Google Scholar). Although the mechanism of action of Sb(V) is still unclear, it is generally believed that Sb(V) must be reduced to Sb(III) to be effective in vivo (6Sereno D. Cavaleyra M. Zemzoumi K. Maquaire S. Ouaissi A. Lemesre J.L. Antimicrob. Agents Chemother. 1998; 42: 3097-3102Google Scholar). Whether Sb(V) is reduced to Sb(III) in Leishmania amastigotes or in host macrophages is controversial. In 2001, Shaked-Mishan et. al. (7Shaked-Mishan P. Ulrich N. Ephros M. Zilberstein D. J. Biol. Chem. 2001; 276: 3971-3976Google Scholar) reported isolating a Pentostam-resistant Leishmania donovani strain that lacks the antimoniate (Sb(V)) reduction, although no enzyme was associated with this activity. Reduction could occur enzymatically as a thiol-dependent reductase found in Leishmania (8Denton H. McGregor J.C. Coombs G.H. Biochem. J. 2004; (in press)Google Scholar), by other types of reductases as in yeast (9Mukhopadhyay R. Shi J. Rosen B.P. J. Biol. Chem. 2000; 275: 21149-21157Google Scholar), or reduction could occur non-enzymatically by intracellular thiols in the parasite or host (10Ferreira Cdos S. Martins P.S. Demicheli C. Brochu C. Ouellette M. Frezard F. Biometals. 2003; 16: 441-446Google Scholar). If reduction occurs in the macrophage then the reduced form Sb(III) would have to be transported into the parasite to be effective. To date, however, no Sb(III) uptake system has been identified in Leishmania. Resistance is an interplay between uptake, efflux, and sequestration. Down-regulation of an uptake system can result in acquired resistance. Aquaglyceroporins (AQPs) 1The abbreviations used are: AQP, aquaglyceroporin; RACE, rapid amplification of cDNA ends; RLM-RACE, RNA ligase-mediated RACE. have recently been identified as uptake systems for trivalent metalloids in eukaryotes and prokaryotes (11Sanders O.I. Rensing C. Kuroda M. Mitra B. Rosen B.P. J. Bacteriol. 1997; 179: 3365-3367Google Scholar, 12Wysocki R. Chery C.C. Wawrzycka D. Van Hulle M. Cornelis R. Thevelein J.M. Tamas M.J. Mol. Microbiol. 2001; 40: 1391-1401Google Scholar, 13Liu Z. Shen J. Carbrey J.M. Mukhopadhyay R. Agre P. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6053-6058Google Scholar). AQPs, which are channels for small neutral solutes, are members of the aquaporin superfamily (14Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Google Scholar). Mammalian AQP9 has the broadest specificity and transports water, glycerol, urea, carbamides, polyols, purines, and pyrimidines in a manner that is sensitive to phloretin and mercury (14Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Google Scholar, 15Tsukaguchi H. Weremowicz S. Morton C.C. Hediger M.A. Am. J. Physiol. 1999; 277: F685-F696Google Scholar). GlpF, an aquaglyceroporin homologue in Escherichia coli, was first identified as an uptake pathway for Sb(III) (11Sanders O.I. Rensing C. Kuroda M. Mitra B. Rosen B.P. J. Bacteriol. 1997; 179: 3365-3367Google Scholar). Disruption of the Saccharomyces cerevisiae FPS1 gene, which encodes an AQP homologue, similarly leads to resistance to trivalent metalloids (12Wysocki R. Chery C.C. Wawrzycka D. Van Hulle M. Cornelis R. Thevelein J.M. Tamas M.J. Mol. Microbiol. 2001; 40: 1391-1401Google Scholar). Recently, we have shown that mammalian AQP7 and AQP9 facilitate uptake of As(III) and Sb(III) when expressed in either S. cerevisiae or oocytes (13Liu Z. Shen J. Carbrey J.M. Mukhopadhyay R. Agre P. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6053-6058Google Scholar). Given that the pKa values for the hydroxides of As(III) and Sb(III) are 9.2 and 11.8, respectively, the anions arsenite and antimonite do not exist in solution at a neutral pH level. We have shown that the species present in solution is As(OH)3 (16Ramirez-Solis A. Mukopadhyay R. Rosen B.P. Stemmler T.L. Inorg. Chem. 2004; 43: 2954-2959Google Scholar), but minor polymeric species probably also exist (17Liu Z. Boles E. Rosen B.P. J. Biol. Chem. 2004; 279: 17312-17318Google Scholar). The purpose of this study was two-fold. First, no aquaglyceroporin homologue has been identified in any Leishmania species. Second, the route of uptake of the activated form of the anti-Leishmania drug Pentostam is unknown. In this study we report for the first time the identification of a leishmanial homologue of the mammalian AQP9 and demonstrate that it is responsible for the uptake of trivalent antimony. LtAQP1 (GenBank™ accession number AY338876) and LmAQP1 (GenBank™ accession number AY567835) were identified from Leishmania tarentolae and Leishmania major, respectively. These exhibit 76% identity and 90% overall similarity to each other. LmAQP1 was expressed in promastigotes from three different species of Leishmania. Each of the transfectants produced substantial amounts of LmAQP1 and became hypersensitive in vitro to As(III) and Sb(III). LmAQP1 expression in a variety of As(III)- or Sb(III)-resistant mutants restored metalloid sensitivity in every strain, independently of the mechanism of resistance. The results of transport studies suggest that this hypersensitivity is caused by an increased rate of uptake of Sb(III) or As(III) in the promastigotes, consistent with increased amounts of the LmAQP1 channel. Disruption of one of the two AQP1 alleles in L. major conferred a 10-fold increase in resistance to trivalent antimony compared with the wild type. These results suggest that a major route of entry of trivalent antimony, the activated form of Pentostam, into Leishmania is catalyzed by AQP1. Importantly, the results demonstrate that a loss of AQP1 can produce resistance and that increased expression of AQP1 in drug-resistant parasites can reverse resistance. Reagents and Media—All reagents were from Sigma. As(III) and Sb(III) were purchased in the form of sodium arsenite and potassium antimonyl tartrate. DNA manipulation reagents were obtained from Qiagen and Invitrogen. Semi-defined medium 79 for growth of Leishmania promastigotes was prepared as described previously (18White T.C. Fase-Fowler F. van Luenen H. Calafat J. Borst P. J. Biol. Chem. 1988; 263: 16977-16983Google Scholar). Cell Lines and Cultures—All species of Leishmania were maintained in Semi-defined medium 79 at 25 °C as promastigotes. For the pathogenic species L. major (LV39) and L. infantum, the medium was supplemented with 10 μm 6-biopterin. The L. tarentolae drug-resistant mutants LtarAs50.1 (selected for resistance to As(III)) and LtarSb400.1 (selected for resistance to Sb(III)) have been described previously (19Haimeur A. Brochu C. Genest P. Papadopoulou B. Ouellette M. Mol. Biochem. Parasitol. 2000; 108: 131-135Google Scholar, 20Ouellette M. Hettema E. Wust D. Fase-Fowler F. Borst P. EMBO J. 1991; 10: 1009-1016Google Scholar). The cell line LinfAs200.1 was selected in the presence of sodium arsenite by in vitro stepwise selection. This cell line grows in the presence of 150 μm arsenite and is cross-resistant to potassium antimonyl tartrate (Table I). All the transfectants were maintained in the presence of 120 μg/ml geneticin (G418, Invitrogen).Table IMetalloid sensitivity of wild type and drug-resistant Leishmania strains transfected with LmAQP1StrainsEC50As(III)Sb(III)μmLtar-neo0.60.2Ltar-AQPneo0.40.1LtarAs50.1-neo80200LtarAs50.1-AQPneo0.50.1LtarSb400.1-neo50>800LtarSb400.1-AQPneo0.50.1Linf-neo25150Linf-AQPneo0.10.06LinfAs200.1-neo1501000LinfAs200.1-AQPneo0.120.4LinfSb4.5-neo400>4000LinfSb4.5-AQPneo0.50.1LV39-neo1.53.0LV39-AQPneo0.10.03LV39Sb600.1-neo40600LV39Sb600.1-AQPneo0.340.1 Open table in a new tab Cloning of Full-length LmAQP1 and LtAQP1—The Leishmania Genome Network gene data was for an L. major homologue of human aquaporin were The homologue to AQP9 is in the was with human or were of that could as as to the of the To the and of the and rapid amplification of cDNA was The was by the a from The cDNA was from of RNA from L. tarentolae a for the provided by the to The was DNA and a and a reverse provided by were in a from The of LtAQP1 cDNA was by the RNA ligase-mediated a from the L. tarentolae RNA was first with to from and DNA for The found of was not by The RNA was then with to the a RNA was to the RNA RNA The could not to RNA the for reverse was virus reverse from by to the of the The was an All were into and from Full-length LtAQP1 could be from and The from LtAQP1 was with that of to the and The DNA from the Leishmania gene data was used to and reverse at the and at the which were used to the from DNA of L. major by The was into and Cloning of LmAQP1 in Leishmania was the of and was as described previously B. Roy G. Ouellette M. EMBO J. Scholar). In this study, the of was into the of to was with and and into the of to Disruption of an LmAQP1 in L. with and of LmAQP1 was from L. major DNA by The was to the of LmAQP1 was with a The containing the was and transfected into wild type L. resistant to geneticin were DNA was with and by J. Scholar), with a DNA of the of LmAQP1 and a one to the against a to of LmAQP1 was by were prepared from Leishmania and L. tarentolae transfected with or LmAQP1 as described previously S. Ouellette M. J. Papadopoulou B. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. Scholar). was M.J. J. Biol. Chem. Scholar). were by Scholar). The proteins were to a and with the at as described previously (9Mukhopadhyay R. Shi J. Rosen B.P. J. Biol. Chem. 2000; 275: 21149-21157Google Scholar). Transfection of LmAQP1 in of into the Leishmania promastigotes was as described previously M. Fase-Fowler F. Borst P. EMBO J. Scholar). were with 120 μg/ml geneticin from Invitrogen. sensitivity of the transfectants and the LmAQP1 strain was as described previously A. Ouellette M. Antimicrob. Agents Chemother. 1998; 42: Scholar). were to in Semi-defined medium 79 containing various of As(III) in the form of sodium arsenite or Sb(III) in the form of potassium antimonyl tartrate. was from the at after was from the of the of metalloid Each was three were from the sensitivity of amastigotes macrophages was by a L. donovani strain, from a from who not to pentavalent antimony with LmAQP1 and gene as described previously G. C. D. Singh M.J. Ouellette M. M. Papadopoulou B. Mol. Biochem. Parasitol. 2000; Scholar). cells were in medium the cells were by treatment with was with medium and then infected with promastigotes AQP1 or at a parasite to macrophage of at °C with for parasites were and infected macrophages were with different of were and of the were with and to a and was in a are expressed as was by the of as a of drug were from the Leishmania promastigotes were with pH and in containing 10 at a of were then with μm As(III) or Sb(III) for 10 at was with the of by at for at with of for at to with water, and then by as described previously C. J. Roy G. N. Ouellette M. Antimicrob. Agents Chemother. 2003; Scholar). Each transport was three with were from the and Cloning of Leishmania Genome Network gene data L. major for aquaporin of the are more to aquaporin channels than to aquaglyceroporins. the in has similarity to aquaglyceroporins. This from L. major, which we has a when with human or To the and of the L. major and the L. tarentolae homologue, and were with RNA from L. were from and respectively, These were and to an gene that we of LtAQP1 with LmAQP1 that the of the from the LmAQP1 was by from L. major DNA as a gene, which was then and found to to the data of LtAQP1 and LmAQP1 with and human AQP9 overall similarity with the mammalian and overall similarity have and the signature motifs of aquaglyceroporins. LmAQP1 was for of LmAQP1 in was in the Leishmania expression and the resulting was transfected into three strains of L. tarentolae, L. infantum, and L. Each strain became hypersensitive to Sb(III) and also to As(III) when transfected with LmAQP1 compared with with (Table I). was in pathogenic strains L. and L. major compared with the strain L. The L. tarentolae strain a increase in sensitivity to As(III) and a increase in sensitivity to Sb(III). the other the L. strain became and more sensitive to As(III) and respectively. The of the L. major strain was for As(III) and for Sb(III). of LmAQP1 in transfected L. tarentolae and L. was detected by with against a to of LmAQP1 and expression of LmAQP1 transport the it is to that this is in that stepwise in vitro, we have promastigotes of strains that are resistant to Sb(III) and As(III) C. J. Roy G. N. Ouellette M. Antimicrob. Agents Chemother. 2003; Scholar, S. Papadopoulou B. Haimeur A. Roy G. Grondin K. D. Rosen B.P. Ouellette M. Mol. Biochem. Parasitol. Scholar). selected against one metalloid were cross-resistant to the other (Table I). when transfected with each resistant became to both metalloids. The of each transfected resistant was than wild type (Table and was to values found in wild type cells transfected with LmAQP1. LmAQP1 of Sb(III) and As(III) in Leishmania of Leishmania AQP1 to transport Sb(III) and As(III) was LmAQP1 transfected strains L. tarentolae L. and L. major rapid uptake of Sb(III) and As(III) compared with transfectants with channels do not to intracellular or in result in an of the for all the Each Leishmania species LmAQP1 rates of uptake and an of As(III) and Sb(III) that was more than the Although the L. tarentolae strain also amounts of As(III) or it was sensitive to the metalloids than other species. This could be of an to the more than described Leishmania species selected for resistance to As(III) or Sb(III) in vitro a reduced of the metalloids compared with the strains C. J. Roy G. N. Ouellette M. Antimicrob. Agents Chemother. 2003; Scholar, S. Papadopoulou B. Haimeur A. Roy G. Grondin K. D. Rosen B.P. Ouellette M. Mol. Biochem. Parasitol. Scholar). resistant mutants were transfected with rates of uptake and of intracellular Sb(III) or As(III) We that the increase in the rate of uptake is responsible for of the resistant mutants to metalloids. This that the uptake of Sb(III) have become for drug action in LmAQP1 of L. field isolate of L. donovani from a who to to pentavalent antimonials in India R. S. Makharia A. R. A. E. D. J. Infect. Dis. 1999; Scholar) and the sensitive strain with as by field were transfected with LmAQP1. strains were more sensitive to Pentostam and as amastigotes compared with the strains transfected with The resistant strain transfected with LmAQP1 was also more sensitive to As(III) or Sb(III) (Table in vitro as promastigotes. These results suggest that uptake be for Pentostam action in as as resistant sensitivity of field Open table in a new tab Disruption of an LmAQP1 Leishmania is a it is to the gene both to a we have a of L. major by of one of the two alleles This is 10-fold more resistant to Sb(III) than the wild type This that the of AQP1 in the is the in uptake of the activated form of the antimonial drugs. also the that mutations in the AQP1 gene could to resistance. This is an it was not a mutation would result in a reduction in uptake to produce resistance. studies the of mutations in the AQP1 gene and the of AQP1 in the of field of drug-resistant We have identified a homologue of mammalian in two strains of Leishmania. Leishmania AQP1 is responsible for As(III) and Sb(III) uptake in Leishmania promastigotes and is the major route of uptake of trivalent metalloids. AQP1 drug sensitivity when expressed at increased in both promastigotes (Table and amastigotes the rate of uptake to such an that it can any mechanism of resistance by the with The of Pentostam and the first line of drug treatment against all forms of is These drugs are in which of the Sb(V) with either (Pentostam) or the increase Sb(V) is by both promastigotes and amastigotes C. J. Roy G. N. Ouellette M. Antimicrob. Agents Chemother. 2003; Scholar), although for pentavalent antimony have not been amastigotes of L. infantum, L. C. J. Roy G. N. Ouellette M. Antimicrob. Agents Chemother. 2003; Scholar), L. Biochem. Physiol. C. Scholar), and L. donovani (7Shaked-Mishan P. Ulrich N. Ephros M. Zilberstein D. J. Biol. Chem. 2001; 276: 3971-3976Google Scholar) more Sb(V) than Sb(III). this an Sb(V) has no against either promastigotes or amastigotes at (6Sereno D. Cavaleyra M. Zemzoumi K. Maquaire S. Ouaissi A. Lemesre J.L. Antimicrob. Agents Chemother. 1998; 42: 3097-3102Google Scholar, J.C. Coombs G.H. Parasitol. Scholar, W.L. Antimicrob. Agents Chemother. Scholar). Pentostam however, were as effective as the against amastigotes of macrophages (6Sereno D. Cavaleyra M. Zemzoumi K. Maquaire S. Ouaissi A. Lemesre J.L. Antimicrob. Agents Chemother. 1998; 42: 3097-3102Google Scholar, D. Roy G. Lemesre J.L. Papadopoulou B. Ouellette M. Antimicrob. Chemother. 2001; Scholar, W.L. Antimicrob. Agents Chemother. Scholar), suggesting that macrophages are in Pentostam activity. We have recently that Sb(III)-resistant amastigotes of L. and L. are cross-resistant to Sb(V) when of Sb(III) is against both forms of the studies found no between and in vitro resistance to Sb(V) with the of the parasite R. S. Makharia A. R. A. E. D. J. Infect. Dis. 1999; however, a was with intracellular parasites R. S. Makharia A. R. A. E. D. J. Infect. Dis. 1999; Scholar). we also a between resistance to Sb(III) and to As(III) in both promastigotes and intracellular amastigotes (Table it is to that a reduction of Sb(V) to Sb(III) is for drug and that of the reduction occurs in host If then expression of amastigote AQP1 be an in drug In amastigotes generally are more sensitive to Sb(III) than promastigotes C. J. Roy G. N. Ouellette M. Antimicrob. Agents Chemother. 2003; Scholar), suggesting that AQP1 could be in the two of the We have that the amastigotes LmAQP1 of macrophages are sensitive to Sb(V) than the promastigotes to Sb(III) (Table One is that the effective of Sb(III) is This could also suggest that the rate of reduction is in the macrophage or that Sb(V) is transported into amastigotes reduction by macrophages and the of Sb(III) in the is however, to that LmAQP1 the resistant strain as sensitive as the wild type. The of this is more than to reverse the drug resistance in the field. Drug resistance is a major impediment to treatment of leishmaniasis (3Guerin P.J. Olliaro P. Nosten F. Druilhe P. Laxminarayan R. Binka F. Kilama W.L. Ford N. White N.J. Lancet Infect. Dis. 2002; 2: 564-573Google Scholar, S. More D.K. Singh M.K. Singh V.P. Sharma S. Makharia A. Kumar P.C. Murray H.W. Clin. Infect. Dis. 2000; 31: 1104-1107Google Scholar). Down-regulation or loss of uptake systems has been shown to to drug resistance (11Sanders O.I. Rensing C. Kuroda M. Mitra B. Rosen B.P. J. Bacteriol. 1997; 179: 3365-3367Google Scholar, 12Wysocki R. Chery C.C. Wawrzycka D. Van Hulle M. Cornelis R. Thevelein J.M. Tamas M.J. Mol. Microbiol. 2001; 40: 1391-1401Google Scholar). results that the loss of one AQP1 allele results in a 10-fold increase in drug resistance. be to drug resistance with loss of AQP1 activity. the expression of AQP1 metalloid resistance in Leishmania strains selected for resistance in vitro or in field The mechanisms of resistance in of strains have been and is in the of resistance C. N. Brochu C. N. A. R. G. D. N. Papadopoulou B. J. Legare D. Ouellette M. 2003; 31: This that expression of AQP1 can reverse resistance in of resistance. We and for in this

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