Skip to main content

Advertisement

SpringerLink
Log in

Frequency of MDR1-related p-gp overexpression in Greek Leishmania isolates

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

An Erratum to this article was published on 19 June 2015

Abstract

In this work, we investigated Greek Leishmania isolates (n = 70) for their individual MDR1-gene-related p-gp (belonging to the ABC-B subfamily of permeases) expression levels by means of flow cytometric analysis of Rhodamine 123 extrusion kinetics. Of all used isolates, 5.71 % express this drug-extruding ABC-transporter at alarming levels and are distributed widely over the country. Some 33 % of all examined isolates originated on the island of Crete though none of the strains showed vastly elevated p-gp extrusion activity, indicating a reasonable implementation of anti-leishmanial compounds in this part of the country. Compared to isolates obtained from canine tissue, human Leishmania isolates were superior both in size and in subcellular differentiation in flow cytometry. Furthermore, a specific t test confirmed verapamil hydrochloride to be a highly potent p-gp reversal agent with p < 0.0001. In a second test series, the loading of Leishmania with Rhodamine 123 was moreover reduced when occurring under influence of verapamil hydrochloride, a known p-gp reversal agent, indicating an ATP-dependant influx of the fluorescent dye and therewith the drug itself. In a final, third experiment series, it was shown that SbV does not act upon the promastigote form of Leishmania.

Comparison of a non - p-gp - expressing (upper graphs) and a p-gp – expressing (lower grpahs) Leishmania isolate: P-gp expression levels of Leishmania with reversed (grey) and non – reversed (blue) MDR 1 – related p-gp transporter activity are objectified by the extrusion of Rhodamine 123, a fluorescent dye and p-gp substrate, over the time (0’ to 120’ mins – from dark to light blue/grey)

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Aït-Oudhia K, Gazanion E, Vergnes B, Oury B, Sereno D (2011) Leishmania antimony resistance: what we know what we can learn from the field. Parasitol Res 109:1225–1232

    Article  PubMed  Google Scholar 

  • Antoniou M, Haralambous C, Mazeris A, Pratlong F, Dedet JP, Soteriadou K (2009) Leishmania donovani leishmaniasis in Cyprus. Lancet Infect Dis 9:76–77

    Article  PubMed  Google Scholar 

  • Ariyanayagam MR, Fairlamb AH (2001) Ovothiol and trypanothione as antioxidants in trypanosomatids. Mol Biochem Parasitol 115:189–198

    Article  CAS  PubMed  Google Scholar 

  • Basselin M, Robert-Gero M (1998) Alterations in membrane fluidity, lipid metabolism, mitochondrial activity, and lipophosphoglycan expression in pentamidine-resistant Leishmania. Parasitol Res 84:78–83

    Article  CAS  PubMed  Google Scholar 

  • Basselin M, Lawrence F, Robert-Gero M (2000) Altered transport properties of pentamidine-resistant Leishmania donovani and L. amazonensis pomastigotes. Parasitol Res 83:413–418

    Article  Google Scholar 

  • Bin Dajem SM, Al–Farsi HM, Al-Hashami ZS, Al-Sheikh AA, Al-Qahtani A, Babiker HA (2012) Distribution of drug resistance genotypes in Plasmodium falciparum in an area of limited parasite diversity in Saudi Arabia. Am J Trop Med Hyg 86:782–788

    Article  PubMed Central  PubMed  Google Scholar 

  • Brochu C, Wang J, Roy G, Messier N, Wang X, Saravia NG, Oullette M (2003) Antimony uptake systems in protozoan parasite Leishmania and accumulation differences in antimony resistant parasites. Antimicrob Agents Chemother 47:3073–3079

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Callahan HL, Roberts WL, Rainey PM, Beverley SM (1994) The PGPA gene of Leishmania major mediates antimony (SbIII) resistance by decreasing influx and not by increasing efflux. Mol Biochem Parasitol 68:145–149

    Article  CAS  PubMed  Google Scholar 

  • Chang G (2003) Multidrug resistance ABC transporters. FEBS Lett 555:102–105

    Article  CAS  PubMed  Google Scholar 

  • Chiquero MJ, Perez-Victoria JM, O’Valle F, González-Ros JM, del Moral RG, Ferragut JA, Castanys S, Gamarro F (1998) Altered drug membrane permeability in a multidrug-resistant Leishmania tropica line. Biochem Pharmacol 55:131–139

    Article  CAS  PubMed  Google Scholar 

  • Chow LM, Wong AK, Ullman B, Wirth DF (1993) Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii. Mol Biochem Parasitol 60:195–208

    Article  CAS  PubMed  Google Scholar 

  • Croft SL, Sundar S, Fairlamb AH (2006) Drug resistance in leishmaniasis. Clin Microbiol Rev 19:111–126

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Dedet JP (2002) Current status of epidemiology of leishmaniasis. In: Farrell JP (ed) World class parasites, vol. 4 Leishmania. Kluwer Academic Publishers, Dordrecht, The Netherlands

    Google Scholar 

  • Desjeux P, Alvar J (2002) Leishmania/HIV co-infections: epidemiology in Europe. Ann Trop Med Parasitol 97:3–15

    Article  Google Scholar 

  • Dweik A, Schoenian G, Mosleh IM, Karanis P (2007) Evaluation of PCR-RFLP (ITS-1 and Hae III) for the detection of Leishmania species using Greek canine isolates and Jordanian clinical material. Ann Trop Med Parasitol 101:399–407

    Article  CAS  PubMed  Google Scholar 

  • Ephros M, Waldman E, Zilberstein D (1997) Pentostam induces resistance to antimony and the preservative chlorocresol in Leishmania donovani promastigotes and axenically grown amastigotes. Antimicrob Agents Chemother 41:1064–1068

    CAS  PubMed Central  PubMed  Google Scholar 

  • Faraut-Gambarelli F, Piarroux R, Deniau M, Giusiano B, Marty P, Michel G, Faugère B, Dumon H (1997) In vitro and in vivo resistance of Leishmania infantum to meglumine antimoniate: a study of 37 strains collected from patients with visceral leishmaniasis. Antimicrob Agents Chemother 41:827–830

    CAS  PubMed Central  PubMed  Google Scholar 

  • Garcia N, Figarella K, Mendoza-Leon A, Ponte-Sucre A (2000) Changes in the infectivity, pyruvate kinase activity, acid phosphatase activity and P-glycoprotein expression in glibenclamide-resistant Leishmania mexicana. Parasitol Res 86:899–904

    Article  CAS  PubMed  Google Scholar 

  • Gourbal B, Sonuc N, Bhattacharjee H, Legaré D, Sundar S, Oullette M, Rosen BP, Mukhopadhyay R (2004) Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin. J Biol Chem 279:31010–31017

    Article  CAS  PubMed  Google Scholar 

  • Gueiros-Filho FJ, Viola JP, Gomes FC, Farina M, Lins U, Bertho AL, Wirth DF, Lopes UG (1995) Leishmania amazonensis: multidrug resistance in vinblastine-resistant promastigotes is associated with rhodamine 123 efflux, DNA amplification, and RNA overexpression of Leishmania mdr1 gene. Exp Parasitol 81:480–490

    Article  CAS  PubMed  Google Scholar 

  • Hadighi R, Boucher P, Khamesipour A, Meamar AR, Roy G, Oullette M, Mohebali M (2007) Glucantime-resistant Leishmania tropica isolated from Iranian patients with cutaneous leishmaniasis are sensitive to alternative anti-leishmanial drugs. Parasitol Res 101:1319–1322

    Article  CAS  PubMed  Google Scholar 

  • Karani M, Sotiriadou I, Plutzer J, Karanis P (2013) Unified loop-mediated isothermal amplification assay for the detection of Leishmania promastigotes. Epidemiol Infect 29:1–7

    Google Scholar 

  • Karanis P, Ongerth J (2009) LAMP—a powerful and flexible tool for monitoring microbial pathogens. Trends Parasitol 25:498–499

    Article  PubMed  Google Scholar 

  • Karanis P, Frank C, Schmale H, Naucke TJ, Jörden U, Metallinou C, Haralabidis S, Maier WA, Seitz HM, Himonas C (2000) Studies on sandfly distribution in Northern Greece. Entomologia Hellenica 3:13–16

    Google Scholar 

  • Katakura K, Fujise H, Takeda K, Kaneko O, Torii M, Suzuki M, Chang KP, Hashiguchi Y (2005) Overexpression of LaMDR2, a novel multidrug resistance ATP - binding cassette transporter, causes 5-fluorouracil resistance in Leishmania amazonensis. FEBS Lett 561:207–212

    Article  Google Scholar 

  • Katsuaki I, Nguyen HT, Kato Y, Wakayama T, Kubo Y, Iseki S, Tsuji A (2008) P-Glycoprotein (Abcb1) is involved in absorptive drug transport in skin. J Control Release 131:198–204

    Article  Google Scholar 

  • Koizumi S, Konishi M, Ichihara T, Wada H, Matsukawa H, Goi K, Mizutani S (1995) Flow cytometric functional analysis of multidrug resistance by fluo-3: a comparison with rhodamine 123. Eur J Cancer 31:1682–1688

    Article  Google Scholar 

  • Kumar D, Singh R, Bhandari V, Kulshrestha A, Negi NS, Salotra P (2012) Biomarkers of antimony resistance: need for expression analysis of multiple genes to distinguish resistance phenotype in clinical isolates of Leishmania donovani. Parasitol Res 111:223–230

    Article  PubMed  Google Scholar 

  • Landfear SM (2008) Drugs and transporters in kinetoplastid protozoa. Adv Exp Med Biol 625:22–32

    Article  CAS  PubMed  Google Scholar 

  • Li Q, Zhao Y, Ni B, Yao C, Zhou Y, Xu W, Wang Z, Qiao Z (2008) Comparison of the expression profiles of promastigotes and axenic amastigotes in Leishmania donovani using serial analysis of gene expression. Parasitol Res 103:821–828

    Article  PubMed  Google Scholar 

  • Liarte DB, Murta SMF (2010) Selection and phenotype characterization of potassium antimony tartrate-resistant populations of four new world Leishmania species. Parasitol Res 107:205–212

    Article  PubMed  Google Scholar 

  • Mandal G, Sarkar A, Saha P, Singh N, Sundar S, Chatterjee M (2009) Functionality of drug efflux pumps in antimonial resistant Leishmanaia donovani field isolates. Indian J Biochem Biophys 46:86–92

    CAS  PubMed  Google Scholar 

  • Mandal S, Maharjan M, Singh S, Chatterjee M, Madhubala R (2010) Assessing aquaglyceroporin gene status and expression profile in antimony-susceptible and -resistant clinical isolates of Leishmania donovani from India. J Antimicrob Chemother 65:496–507

    Article  CAS  PubMed  Google Scholar 

  • Marquis N, Gourbal B, Rosen BP, Mukhopadhyay R, Oullette M (2005) Modulation in aquaglyceroporin AQP1 gene transcript levels in drug-resistant Leishmania. Mol Microbiol 57:1690–1699

    Article  CAS  PubMed  Google Scholar 

  • Mary C, Faraut F, Lascombe L, Dumon H (2004) Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol 42:5249–5255

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mary C, Faraut F, Deniau M, Dereure J, Aoun K, Ranque S, Piarroux R (2010) Frequency of drug resistance gene amplification in clinical Leishmania strains. Int J Microbiol. doi:10.1155/2010/819060

    PubMed Central  PubMed  Google Scholar 

  • Mittal MK, Rai S, Ravinder A, Gupta S, Sundar S, Goyal N (2007) Characterization of natural antimony resistance in Leishmania donovani isolates. Am J Trop Med Hyg 76:681–688

    CAS  PubMed  Google Scholar 

  • Nogueira YL, Nakamura PM, Galati EAB (2006) Kinetics of growth of Leishmania chagasi cycle in McCoy cell culture. Rev Inst Med Trop 48:337–341

    Article  Google Scholar 

  • Pineda JA, Martín-Sánachez J, Macías J, Morillas F (2002) Leishmania spp. infection in injecting drug users. Lancet 360:950–951

    Article  PubMed  Google Scholar 

  • Pral EMF, Moitinho MLR, Balanco JMF, Teixeira VR, Milder RV, Alfieri SC (2003) Growth phase and medium pH modulate the expression of proteinase activities and the development of megasomes in axenically cultivated Leishmania amazonensis amastigote-like organisms. J Parasitol 89:35–43

    Article  CAS  PubMed  Google Scholar 

  • Prasad V, Kaur J, Dey CS (2000) Arsenite-resistant Leishmania donovani promastigotes express an enhanced membrane P-type adenosine triphosphatase activity that is sensitive to verapamil treatment. Parasitol Res 86:661–664

    Article  CAS  PubMed  Google Scholar 

  • Purkait B, Kumar A, Nandi N, Sardar AH, Das S, Kumar S, Pandey K, Ravidas V, Kumar M, De T, Singh D, Das P (2012) Mechanisms of amphotericin B resistance in clinical isolates of Leishmania donovani. Antimicrob Agents Chemother 56:1031–1041

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ready PD (2010) Leishmaniasis emergence in Europe. Euro Surveill 15:1–11

    Google Scholar 

  • Romsicki Y, Sharom FJ (2001) Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter. Biochemistry 40:6937–6947

    Article  CAS  PubMed  Google Scholar 

  • Shapiro AB, Ling V (1998) The mechanism of ATP-dependant multidrug transport by P-glycoprotein. Acta Physiol Scand Suppl 643:227–234

    CAS  PubMed  Google Scholar 

  • Sinha PK, Bhattacharya SK (2011) Antimicrobial resistance in Leishmania donovani in Bihar, India. Reg Health Forum 15:134

    Google Scholar 

  • Tang F, Ouyang H, Yang JZ, Borchardt RT (2004) Bidirectional transport of Rhodamine 123 and Hoechst 33342, fluorescence probes of the binding sites on P-glycoprotein, across MDCK-MDR1 cell monolayers. J Pharm Sci 93:1185–1194

    Article  CAS  PubMed  Google Scholar 

  • Trotz-Williams LA, Trees AJ (2003) Systematic review of the distribution of the major vector-borne parasitic infections in dogs and cats in Europe. Vet Rec 152:97–105

    Article  CAS  PubMed  Google Scholar 

  • Troutman MD, Thakker DR (2003) Rhodamine 123 requires carrier-mediated influx for its activity as a P-glycoprotein substrate in Caco-2 Cells. Pharm Res 20:1192–1199

    Article  CAS  PubMed  Google Scholar 

  • Vanerschoot M, Maes I, Ouakad M, Adaui V, Maes L, De Doncker S, Rijal S, Chappuis F, Dujardin JC, Decuypere S (2010) Linking in vitro and in vivo survival in clinical Leishmania donovani strains. PLOS One 5:e12211

    Article  Google Scholar 

  • Varadi A, Szakács G, Bakos E, Sarkadi B (2002) P glycoprotein and the mechanism of multidrug resistance. Novartis Found Symp 243:54–65

    Article  CAS  PubMed  Google Scholar 

  • Warburg A, Gelman S, Deutsch J (2008) Xanthine in urine stimulates growth of Leishmania promastigotes in vitro. J Med Microbiol 57(pt1):136–138

    Article  CAS  PubMed  Google Scholar 

  • WHO (2000) Leishmania/HIV co-infection in south-western Europe 1990–1998: Retrospective analysis of 965 cases. WHO/LEISH/2000.42

  • Wong IL, Chow LM (2006) The role of Leishmania enriettii multidrug resistance protein 1 (LeMDR1) in mediating drug resistance is iron-dependent. Mol Biochem Parasitol 150:278–287

    Article  CAS  PubMed  Google Scholar 

  • Wong IL, Chan KF, Burkett BA, Zhao Y, Chai Y, Sun H, Chan TH, Chow LM (2007) Flavonoid dimers as bivalent modulators for pentamidine and sodium stibogluconate resistance in Leishmania. Antimicrob Agents Chemother 51:930–940

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

We wish to thank former PhD students Ippokrates Messaritakis, Panthelis Ntais and Vassiliki Christodoulou for the scientific exchange in the matter of laboratory practices. Eleni Koutala, technical assistant of the haematology department (University Hospital of Heraklion, Crete), kindly introduced us to the flow cytometer. Finally, we want to thank IMSIE (Institute of Medical Statistics, Informatics and Epidemiology, University of Cologne) for their kind help with the mathematical interpretation of the achieved data.

Author information

Authors and Affiliations

  1. Center of Anatomy, Institute II, Laboratory for Medical and Molecular Parasitology, Medical School, University of Cologne, Cologne, Germany

    Johannes Austrup & Panagiotis Karanis

  2. National Research Center for Protozoan Diseases, Obihiro University for Agriculture and Veterinary Medicine, Hokkaido, Japan

    Panagiotis Karanis

Authors
  1. Johannes Austrup
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Panagiotis Karanis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Johannes Austrup.

Additional information

This work is a part of a doctoral medical thesis at the University of Cologne, Medical School. The experimental part was conducted at the Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine at the University of Crete (UoC, Heraklion, Greece) with the support of Ass. Prof. M. Antoniou. Former PhD student and practicing veterinarian Panthelis Ntais kindly provided the Leishmania isolates for the experiments.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Austrup, J., Karanis, P. Frequency of MDR1-related p-gp overexpression in Greek Leishmania isolates. Parasitol Res 113, 1225–1232 (2014). https://doi.org/10.1007/s00436-014-3761-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-014-3761-8

Keywords

Search

Navigation