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.

Figure 1. Rhod-123 efflux in Leishmania promastigotes observed by Flow Cytometry.

High Rhod-123 efflux in the “resistant” dog isolate D5 (a). Low Rhod-123 efflux in the “susceptible” dog isolate D1 (b). Measurements were taken every 30 minutes, for two hours.

Figure 2. Rate of efflux of Rhod-123, in the 10 Leishmania isolates, estimated by Flow Cytometry.

Efflux, both in the absence (slope “α”) and presence (slope “β”) of Verapamil hydrochloride was found to be correlated to the influx (the amount of drug that entered the parasite body: Mean Fluorescent Intensity on baseline (MFI) (c = 0.63 and c = 0.58, respectively). Efflux was blocked by Verapamil hydrochloride in all isolates except in isolate D2, with the lowest slope “α”, for which slope “β” had the same value (0.16)*.

Figure 3. Detection of Pgp 170 by Western Blotting in the 10 Leishmania isolates.

The Pgp 170 molecules (130-200 kDa) were evident in the “resistant” (high Rhod-123 efflux) but not in the “susceptible” (low Rhod-123 efflux) isolates. The “resistant” human isolate (H2) presented lower signal compared to the dog “resistant” isolates. For the evaluation of the Pgp expression in each isolate, the C219 monoclonal antibody and exponential phase promastigotes, at a concentration of 10⁷ parasites, were used.

Figure 4. Treatment with Glucantime resulted in an increase in the number of Pgp molecules.

Mean Fluorescent Intensity (MFI) of the 10 Leishmania isolates, measured by Flow Cytometry, showed an increase in the number of Pgp molecules, in all isolates, if they had previously been treated with the drug Glucantime compared to the untreated controls. This increase was not linear (r₂ = 0.27).

Figure 5. Mean Fluorescent Intensity (MFI) before and after treatment with Glucantime.

In all 10 isolates, irrespective of the rate of Rhod-123 efflux (slope “α”), treatment with Glucantime resulted in an increase of MFI, estimated by Confocal Microscopy (CM), indicating an increase in the number of Pgp 170 molecules.

Figure 6. Location of Pgp molecules in the body of Leishmania observed under Confocal Microscopy.

Leishmania amastigotes (inside THP-1 infected cells) marked with double immunofluorescence: positive dog serum and FITC (green signal) (a); Pgp 170 molecules marked with the C219 monoclonal antibody and the infrared Cy3 anti-mouse IgG secondary antibody (red signal) (b); overlay of image a and b (c). Image c confirms the location of Pgp 170 molecules on the membrane of the parasite body.

Figure 7. Detection of Pgp 170 molecules, by Transmission Electron Microscopy, after immunogold labeling.

Leishmania amastigote (inside a THP-1 infected cell): cytoplasm of the THP-1 cell (a); Leishmania infantum body (b). Black spots (as indicated by white arrow) show Pgp 170 molecules (gold granules after immunogold labeling using the C219 monoclonal antibody).

Figure 8. Number of Pgp molecules in a ''resistant'' and a ''susceptible'' isolate before/after exposure to Glucantime.

The number of Pgp 170 molecules was found in higher numbers in the “resistant” compared to the “susceptible” isolate. This number increased, in both isolates, after exposure to Glucantime.

Figure 9. Survival of Leishmania promastigotes in different Amphotericin B concentrations, in time.

Survival of the “resistant” dog isolate D5 (a) and of the “susceptible” dog isolate D1 (b), in different drug concentrations for 6 days.