Drug Resistance in Natural Isolates of Leishmania donovani s.l. Promastigotes Is Dependent of Pgp170 Expression

Abstract

Resistance of pathogens to drugs is a growing concern regarding many diseases. Parasites like Leishmania, Plasmodium and Entamoeba histolytica; and neoplastic cells, present the multidrug-resistant phenotype rendering chemotherapy ineffective. The acquired resistance of Leishmania to antimony has generated intense research on the mechanisms involved but the question has not yet been resolved. To test the hypothesis that drug efflux in Leishmania, as measured by flow cytometry using the fluorescent dye Rhodamine-123, is largely dependent on the number of efflux pumps an isolate can express, the amount of Pgp 170 molecules was assessed in ten field isolates (5 “resistant” and 5 “susceptible”) using: Western Blotting, Confocal and Transmission Electron Microscopy, and proteomics. Their survival after exposure to three antileishmanial drugs, in vitro, was evaluated and clinical data were compared to the in vitro results. All isolates were resistant to Glucantime but susceptible to Miltefosine, whilst Amphotericin B was more effective on the “susceptible” isolates. The MDR gene, expressing the transmembrane efflux pump Pgp 170, appears to play a key role in the phenomenon of drug resistance. When “susceptible” versus “resistant” parasites were compared, it was shown that the higher the number of Pgp 170 molecules the higher the Rhodamine-123 efflux from the parasite body and, when exposed to the drug, the number of efflux pumps increased. However, the rate of this increase was not linear and it is possible that there is a maximum number of Pgp 170 molecules an isolate can express. Nevertheless, the phenomenon is a complex one and other factors and proteins are involved in which the HSP-70 group proteins, detected in the “resistant” isolates, may play a significant role.

Geographical Distribution of MDR1 Expression in Leishmania Isolates, from Greece and Cyprus, Measured by the Rhodamine-123 Efflux Potential of the Isolates, Using Flow Cytometry

“…Messaritakis and others 18 related the efflux rate of Leishmania isolates to the number of Pgp molecules, using the C219 monoclonal antibody, and found them, consistently, to be higher in the isolates with a higher rate of efflux, as shown by FCM. This was also true for the isolates if they were exposed to Glucantime as a result of which, the number of pumps as well as the efflux rate of the isolates, increased.…”

“…16 It has been shown that Leishmania, under arsenite and methotrexate selection, overamplifies and expresses genes (e.g., ItpgpA) that are part of the gene family encoding the Pgp. 18,[36][37][38] A member of this gene family (ldmdr1) can induce a drug-resistant phenotype in Leishmania 39 and has been molecularly characterized in L. donovani.…”

“…The rate of efflux of the fluorescent probe Rhod-123 (SigmaAldrich Inc., St. Louis, MO) was tested in all 275 isolates with and without verapamil hydrochloride (blocker of MDR to check the specificity of the pumps) using FCM (Beckman Coulter Epics Elite Flow Cytometer, equipped with argon-ion laser tuned to 630 nm), as described in Messaritakis and others. 18 The mean fluorescence intensity (MFI) of 10,000 events was measured every 30 minutes, for 2 hours, in triplicates, to follow the rate of Rhod-123 efflux for each isolate. Measurements were considered only if the percentage of dead parasites in a run was < 5%.…”

“…[12][13][14][15] The multidrug resistance (MDR1) gene expresses a transmembrane efflux pump (the P-glycoprotein molecule, Pgp 170 [16][17][18] ), which appears to play a key role in the phenomenon of drug resistance. 15,18 Control of the disease relies principally on chemotherapy, both in patients and dogs, 19 and antimony has been the main drug to combat the disease in the last 65 years. Antimony-resistant Leishmania strains have been reported from many endemic areas, worldwide, reaching epidemic proportions in the state of Bihar, India 20,21 and the phenomenon appears to be intensifying and geographically spreading.…”

“…15,18 This pump is effective for a diverse group of lipophilic compounds [15][16][17] and the higher the number of the Pgp 170 molecules on the cell membrane, the greater the ability of the isolate to expel the compound from its body. 14,18,[22][23][24] Our aim was to evaluate the MDR1 expression of Leishmania in Greece and Cyprus and present it geographically to assess the distribution of drug resistance and give the clinicians the knowledge required for effective treatment, which will safeguard against the expansion of this phenomenon. For this purpose, the rate of efflux of Rhod-123 of 211 Leishmania strains isolated from patients and dogs in Greece and of 64 strains isolated from patients and dogs in Cyprus were evaluated using FCM.…”