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. 2015 Apr 7;9(4):e0003704.
doi: 10.1371/journal.pntd.0003704. eCollection 2015 Apr.

Fitness of Leishmania donovani parasites resistant to drug combinations

Raquel García-Hernández  1 , Verónica Gómez-Pérez  1 , Santiago Castanys  1 , Francisco Gamarro  1
Affiliations

Fitness of Leishmania donovani parasites resistant to drug combinations

Raquel García-Hernández et al. PLoS Negl Trop Dis. .

Abstract

Drug resistance represents one of the main problems for the use of chemotherapy to treat leishmaniasis. Additionally, it could provide some advantages to Leishmania parasites, such as a higher capacity to survive in stress conditions. In this work, in mixed populations of Leishmania donovani parasites, we have analyzed whether experimentally resistant lines to one or two combined anti-leishmanial drugs better support the stress conditions than a susceptible line expressing luciferase (Luc line). In the absence of stress, none of the Leishmania lines showed growth advantage relative to the other when mixed at a 1:1 parasite ratio. However, when promastigotes from resistant lines and the Luc line were mixed and exposed to different stresses, we observed that the resistant lines are more tolerant of different stress conditions: nutrient starvation and heat shock-pH stress. Further to this, we observed that intracellular amastigotes from resistant lines present a higher capacity to survive inside the macrophages than those of the control line. These results suggest that resistant parasites acquire an overall fitness increase and that resistance to drug combinations presents significant differences in their fitness capacity versus single-drug resistant parasites, particularly in intracellular amastigotes. These results contribute to the assessment of the possible impact of drug resistance on leishmaniasis control programs.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Growth profile of mixed promastigotes populations of Leishmania lines.
(A) Log-phase promastigotes from WT and resistant lines were mixed at a 1:1 parasite ratio (1x106 parasites/ml) with log-phase promastigotes from Luc line (Luc), and the number of parasites were monitored every 24 h. (B) Luminescence of 107 promastigotes from mixed populations was determined after 6 days of incubation in standard culture medium. Results are the average of three independent experiments ± SD. Horizontal line indicates the threshold at which luminescence levels have been modified with respect to control (WT+Luc).
Fig 2
Fig 2. Leishmania resistant lines are more tolerant to stress in the late stationary growth phase.
Promastigotes from WT and resistant lines were mixed at a 1:1 parasite ratio with promastigotes from Luc line (Luc), maintained in standard culture medium for 7 days and the luminescence of 107 promastigotes was determined at 48 h of the first (black columns) and second (gray columns) sub-culture cycle. Results are the average of four independent experiments ± SD. Statistically differences using Student's t test are indicated relative to the control values (*, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.001). Horizontal line indicates the threshold at which luminescence levels have been modified with respect to control (WT+Luc).
Fig 3
Fig 3. Leishmania resistant lines show higher survival in starvation.
Promastigotes from WT and resistant Leishmania lines were mixed at a 1:1 parasite ratio with promastigotes from Luc line (Luc), incubated in serum-free medium for 24 h, supplemented with 20% hiFBS and the luminescence of 107 promastigotes was determined at 48 h of the first (black columns) and second (gray columns) starvation cycle. Results are the average of four independent experiments ± SD. Statistically differences using Student's t test are showed relative to the control values (*, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.001). Horizontal line indicates the threshold at which luminescence levels have been modified with respect to control (WT+Luc).
Fig 4
Fig 4. Leishmania resistant lines are more tolerant to heat shock and pH stress.
Mixed promastigotes from WT and resistant lines at a 1:1 parasite ratio were incubated in culture medium, pH 5.4 at 37 ºC for 24 h. Then, mixed populations were incubated at 28 ºC and pH 7.2 and luminescence of 107 promastigotes was evaluated at the first (black columns) and second (gray columns) stress cycle. Results are the average of four independent experiments ± SD. Statistically differences using Student's t test are showed relative to the control values (*, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.001). Horizontal line indicates the threshold at which luminescence levels have been modified with respect to control (WT+Luc).
Fig 5
Fig 5. Intracellular amastigotes of Leishmania resistant lines present higher capacity to survive in mouse peritoneal macrophages.
Mouse peritoneal macrophages were plated in 96-well white polystyrene microplates and infected during 6 h with promastigotes of 6 days of culture from resistant or WT lines mixed with Luc line. Infected macrophages were maintained in culture medium at 37°C with 5% CO2 during 24 h (A) or 96 h (B) and luminescence was determined and normalized by infection index. Results are the average of three independent experiments ± SD. Statistically differences using Student's t test are showed relative to the control values (*, p<0.05; **, p<0.01; ***,p<0.001). Horizontal line indicates the threshold at which luminescence levels have been modified with respect to control (WT+Luc).
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Grants and funding

This work was supported by Plan Andaluz de Investigación (Proyecto de Excelencia CTS-7282), Junta de Andalucia (FG). Spanish Grants SAF2012-34267 (to FG) and SAF2011-28102 (to SC). Subdirección General de Redes y Centros de Investigación Cooperativa-FEDER, RICET project RD12/0018/0017 (FG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.