Host cholesterol influences the activity of sterol biosynthesis inhibitors in <i>Leishmania amazonensis</i>

A significant percentage of exogenous cholesterol was found in promastigotes and amastigotes of all studied species of Leishmania, suggesting a biological role for this molecule. Previous studies have shown that promastigotes of Leishmania uptake more low-density lipoprotein (LDL) particles under pharmacological pressure and are more susceptible to ergosterol inhibition in the absence of exogenous sources of cholesterol. This work shows that the host’s LDL is available to intracellular amastigotes and that the absence of exogenous cholesterol enhances the potency of sterol biosynthesis inhibitors in infected macrophages. A complete understanding of cholesterol transport to the parasitophorous vacuole can guide the development of a new drug class to be used in combination with sterol biosynthesis inhibitors for the treatment of leishmaniases.

Key words:
leishmaniasis; low-density lipoprotein; cholesterol; sterol biosynthesis inhibitors

Authorship

Valter Viana Andrade-Neto

Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Bioquímica de Tripanossomatídeos, Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz-FiocruzBrazilRio de Janeiro, RJ, BrazilFundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Bioquímica de Tripanossomatídeos, Rio de Janeiro, RJ, Brasil

Pedro Paulo de Abreu Manso

Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Patologia, Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz-FiocruzBrazilRio de Janeiro, RJ, BrazilFundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Patologia, Rio de Janeiro, RJ, Brasil

Miria Gomes Pereira

Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Laboratório de Ultraestrutura Celular Hertha Meyer, Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de JaneiroBrazilRio de Janeiro, RJ, BrazilUniversidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Laboratório de Ultraestrutura Celular Hertha Meyer, Rio de Janeiro, RJ, Brasil

Nuccia Nicole Theodoro de Cicco

Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de JaneiroBrazilRio de Janeiro, RJ, BrazilUniversidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Rio de Janeiro, RJ, Brasil

Georgia Corrêa Atella

Marcelo Pelajo-Machado

Rubem Figueiredo Sadok Menna-Barreto

Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Biologia Celular, Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz-FiocruzBrazilRio de Janeiro, RJ, BrazilFundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Biologia Celular, Rio de Janeiro, RJ, Brasil

Eduardo Caio Torres-Santos ⁺+ Corresponding author: ects@ioc.fiocruz.br

http://orcid.org/0000-0003-2240-4519

+ Corresponding author: ects@ioc.fiocruz.br

VVAN and ECTS - Conceptualisation; VVAN, PPAM, MGP and NNTC - data curation; GCA, MPM, RFSMB and ECTS - formal analysis; VVAN, PPAM, MGP, RFSMB and ECTS - investigation; VVAN, PPAM, MGP, GCA, MPM, RFSMB and ECTS - methodology; all authors - writing - review and editing.

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Figures

Figures (3)

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Fig. 1:
distribution of low-density lipoprotein (LDL) particles in macrophages infected with Leishmania amazonensis-GFP. Peritoneal macrophages infected with L. amazonensis-GFP were incubated with LDL-Alexa 594 particles for 30 (A and B) or 150 min (C - D) at concentrations of 0.125 (A and C) and 0.5 µg/mL (B and D). After this period, the cells were washed three times in phosphate buffered saline (PBS), fixed with 4% formaldehyde for 10 min and incubated with DAPI for 20 min. Slides were mounted using Prolong^® Gold Antifade Mounting Fluid (Invitrogen, Life Technologies Corporation) and analysed under a Zeiss LSM 510 META confocal microscope. Green: L. amazonensis amastigotes; red: LDL particles; blue: nuclei/kinetoplasts (DAPI). Bars: A = 50 µm; B-D = 20 µm.

Thumbnail

Fig. 2:
availability of low-density lipoprotein (LDL)-gold to intracellular amastigotes. Peritoneal macrophages infected or not with Leishmania amazonensis were incubated with LDL-gold (10 nm) for 4 h and analysed by transmission electron microscopy. A and B: uninfected macrophages; C and D: infected macrophages. Arrows: LDL particles; asterisks: L. amazonensis amastigotes; VP: parasitophorous vacuole. The asterisk represents the amastigotes within the parasitophorous vacuole. Bars = 0.5 µm.

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Fig. 3:
effect of the absence of exogenous cholesterol on the leishmanicidal activity of ketoconazole and miconazole. Peritoneal macrophages infected with Leishmania amazonensis were incubated in RPMI culture medium supplemented with foetal bovine serum (FBS) or cholesterol-free Nutridoma. Cultures remained untreated or were treated with ketoconazole (A) or miconazole (B) for 72 h. The experiments were carried out in triplicate, n = 3. The graphs are representative of an experiment with the standard deviation value. The graphs and IC₅₀ values were obtained using the GraphPad Prism 7 program. Student’s t test, *p < 0.5, **p > 0.001.

Fig. 1: distribution of low-density lipoprotein (LDL) particles in macrophages infected with Leishmania amazonensis-GFP. Peritoneal macrophages infected with L. amazonensis-GFP were incubated with LDL-Alexa 594 particles for 30 (A and B) or 150 min (C - D) at concentrations of 0.125 (A and C) and 0.5 µg/mL (B and D). After this period, the cells were washed three times in phosphate buffered saline (PBS), fixed with 4% formaldehyde for 10 min and incubated with DAPI for 20 min. Slides were mounted using Prolong^® Gold Antifade Mounting Fluid (Invitrogen, Life Technologies Corporation) and analysed under a Zeiss LSM 510 META confocal microscope. Green: L. amazonensis amastigotes; red: LDL particles; blue: nuclei/kinetoplasts (DAPI). Bars: A = 50 µm; B-D = 20 µm.

Fig. 2: availability of low-density lipoprotein (LDL)-gold to intracellular amastigotes. Peritoneal macrophages infected or not with Leishmania amazonensis were incubated with LDL-gold (10 nm) for 4 h and analysed by transmission electron microscopy. A and B: uninfected macrophages; C and D: infected macrophages. Arrows: LDL particles; asterisks: L. amazonensis amastigotes; VP: parasitophorous vacuole. The asterisk represents the amastigotes within the parasitophorous vacuole. Bars = 0.5 µm.

Fig. 3: effect of the absence of exogenous cholesterol on the leishmanicidal activity of ketoconazole and miconazole. Peritoneal macrophages infected with Leishmania amazonensis were incubated in RPMI culture medium supplemented with foetal bovine serum (FBS) or cholesterol-free Nutridoma. Cultures remained untreated or were treated with ketoconazole (A) or miconazole (B) for 72 h. The experiments were carried out in triplicate, n = 3. The graphs are representative of an experiment with the standard deviation value. The graphs and IC₅₀ values were obtained using the GraphPad Prism 7 program. Student’s t test, *p < 0.5, **p > 0.001.

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Host cholesterol influences the activity of sterol biosynthesis inhibitors in <i>Leishmania amazonensis</i>

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[1] + Corresponding author: ects@ioc.fiocruz.br