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. 2002 Feb;46(2):487-99.
doi: 10.1128/AAC.46.2.487-499.2002.

Ultrastructural and biochemical alterations induced by 22,26-azasterol, a delta(24(25))-sterol methyltransferase inhibitor, on promastigote and amastigote forms of Leishmania amazonensis

Juliany C F Rodrigues  1 , Márcia Attias, Carlos Rodriguez, Julio A Urbina, Wanderley de Souza
Affiliations

Ultrastructural and biochemical alterations induced by 22,26-azasterol, a delta(24(25))-sterol methyltransferase inhibitor, on promastigote and amastigote forms of Leishmania amazonensis

Juliany C F Rodrigues et al. Antimicrob Agents Chemother. 2002 Feb.

Abstract

We report on the antiproliferative effects and the ultrastructural and biochemical alterations induced in vitro by 22,26-azasterol, a sterol Delta(24(25))-methyltransferase (24-SMT) inhibitor, on Leishmania amazonensis. When promastigotes and amastigotes were exposed to 100 nM 22,26-azasterol, complete growth arrest and cell lysis ensued after 72 (promastigotes) or 120 (amastigotes) h. Exposure of parasites to this azasterol led to the complete depletion of parasite endogenous sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (zymosterol, cholesta-5,7,24-trien-3beta-ol, and cholesta-7,24-dien-3beta-ol), while 14-methyl-zymosterol and 4,14-dimethyl-zymosterol accumulated as a result of simultaneous incubation of the parasites with 22,26-azasterol and ketoconazole, a known inhibitor of the parasite's sterol C14-demethylase. These results confirmed that 24-SMT is the primary site of action of the azasterol. Profound changes were also observed in the phospholipid compositions of treated cells, in which a twofold reduction in the content of phosphatidylserine was observed; this was accompanied by a concomitant increase in the content of phosphatidylinositol. Transmission electron microscopy showed that 22,26-azasterol induced marked morphological changes, including mitochondrial swelling, invaginations of the inner mitochondrial membrane, and the appearance of large bodies containing concentric membranes. Other modifications included increases in the numbers of acidocalcisomes, megasomes, and lipid inclusions and the appearance of typical autophagic structures and cell body protrusions toward the flagellar pocket. We conclude that the dramatic alteration of the lipid composition of the parasite's membranes induced by the drug underlies the ultrastructural alterations that lead to the loss of cell viability and that 24-SMT inhibitors could be useful as selective antileishmanial agents.

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Figures

FIG. 1.
FIG. 1.
Molecular structure of 22,26-azasterol. Me, methyl.
FIG. 2.
FIG. 2.
Effect of azasterol on the growth of L. amazonensis promastigotes (A) and amastigotes (B). Parasites of both forms were incubated as described in Materials and Methods, and the drug was added to the cultures at 0 h. The cells were counted daily. ⧫, control; ▪, DMSO-treated control; ▴, treatment with 0.1 μM azasterol; * (top), treatment with 1.0 μM azasterol; * (bottom), treatment with 10 μM azasterol. The bars indicate standard deviations.
FIG. 3.
FIG. 3.
Minimum model for the sterol biosynthesis pathway in L. amazonensis promastigotes. I, squalene; II, lanosterol; III, 4,14-dimethyl-zymosterol; IV, 14-methyl-zymosterol; V, 4,14-dimethyl-fecosterol; VI, 14-methyl-fecosterol; VII, cholesta-8,24-dien-3β-ol (zymosterol); VIII, cholesta-5,8,24-trien-3β-ol; IX, cholesta-7,24-dien-3β-ol; X, cholesta-5,7,24-trien-3β-ol; XI, ergosta-7,24(241)-dien-3β-ol (episterol); XII, ergosta-5,7,24(241)-trien-3β-ol (5-dehydroepisterol); XIII, ergosterol. SQSI, squalene synthase inhibitors; SEI, squalene epoxidase inhibitors; SMTI, sterol methyltransferase inhibitors; SMRI, sterol methylreductase inhibitors; SDMI, sterol 14-demethylase inhibitors. See text for details.
Figure 4-6
Figure 4-6
FIG. 4–5: Electron micrographs of promastigote and amastigote forms of L. amazonensis showing a normal morphology in the presence of 0.1% DMSO, the same concentration of DMSO as in the final solutions of azasterol used. N, nucleus; K, kinetoplast; FP, flagellar pocket; M, mitochondrion; GC, Golgi complex. Bars, 1 μm. FIG. 6: Promastigote treated with 1.0 μM azasterol for 24 h. This parasite presented a protrusion of the cell body toward the flagellar pocket (arrow). Bar, 0.5 μm.
FIG. 7–9.
FIG. 7–9.
Protrusions of the flagellar pocket in sequence. The promastigote form was treated with 1.0 μM azasterol for 24 h. The arrows show the protrusions formed by the membrane, and the stars show the vesicles released from the cell body into the flagellar pocket. FP, flagellar pocket. Bars, 1.0 μm.
FIG. 10–13.
FIG. 10–13.
Amastigote forms treated with 0.1 μM azasterol for 72 h (Fig. 10) and 1.0 μM azasterol for 48 h (Fig. 11) presented myelin-like figures within flagellar pocket (arrows). In Fig. 11 we observed a megasome (asterisk). N, nucleus; K, kinetoplast; FP, flagellar pocket. Bars, 0.5 μm.
Figure 14-17
Figure 14-17
FIG. 14–15: Promastigotes treated with 10 μM azasterol for 48 h (Fig. 14; stars) and 1.0 μM azasterol for 48 h (Fig. 15; asterisks) presented intense mitochondrial swelling. N, nucleus. Bars, 1 μm (Fig. 14) and 0.5 μm (Fig. 15). FIG. 16: Electron micrograph showing increases in the numbers of acidocalcisomes (arrowheads) in association with the endoplasmic reticulum. Bar, 0.5 μm. FIG. 17: Ultrathin section of promastigote form treated with azasterol showing the mitochondrion enveloping by endoplasmic reticulum (arrows). N, nucleus. Bar, 0.5 μm.
FIG. 18–20.
FIG. 18–20.
Three-dimensional reconstruction images showing the control promastigote form presenting a normal morphology in the mitochondria. Gray, plasma membrane; blue, nucleus; green, mitochondria; pink, kinetoplast; brown, Golgi complex. We did not observe concentric membranes in the mitochondrial matrix in control cells.
Figure 21-22
Figure 21-22
FIG. 21-22. Promastigote treated with 10 μM azasterol for 24 h presented a myelin-like figure in the cytoplasm (arrows). Bar, 0.5 μm. Ultrathin section of promastigote treated with 0.1 μM azasterol for 48 h showing proliferation in the number of autophagosomal structures (asterisks). Bar, 0.5 μm.
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