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.
Highlights
Leishmania parasites are transmitted by Phlebotomine sandflies causing leishmaniasis
Parasites Seventy Leishmania strains isolated from patients (5, L. donovani) and dogs (65, L. infantum) during an epidemiological survey conducted in Cyprus [5], were used in Flow Cytometry (FCM) experiments to study the rate of efflux, of the fluorescent probe Rhod-123, from the parasite body
Analysis by Western Blotting (WB) The P-glycoprotein hybridization signal was amplified only in the 5 isolates characterized by FCM as ‘‘resistant’’
Summary
Leishmania parasites are transmitted by Phlebotomine sandflies causing leishmaniasis. Leishmania donovani donovani and L. donovani infantum are mainly responsible for visceral leishmaniasis (VL), the most severe form of the disease. In southern Europe VL is endemic due to L. infantum [1] but in the last years L. donovani has emerged in Cyprus both in the cutaneous and visceral form [2]. Whilst L. infantum is zoonotic, using the dog as reservoir host, L. donovani is considered anthroponotic; and as the two parasites meet in the host (vector and reservoir), there is danger of hybrid development with possible new characteristics, unfavourable to the patient [3]. Antimony-resistant Leishmania parasites have been reported from many endemic areas worldwide reaching epidemic proportions in the state of Bihar, India [7,8]
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