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Antimicrobial Agents and Chemotherapy, February 2002, p. 487-499, Vol. 46, No. 2
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.2.487-499.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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,1 Carlos Rodriguez,2 Julio A. Urbina,2* and Wanderley de Souza1*

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco G, Ilha do Fundão, 21949-900, Brazil,1 Laboratório de Química Biológica, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas, Apartado 21827, Caracas 1020A, Venezuela2

Received 2 April 2001/ Returned for modification 3 July 2001/ Accepted 30 October 2001

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-3ß-ol, and cholesta-7,24-dien-3ß-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.

* Mailing address for Julio A. Urbina: Laboratório de Química Biológica, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas, Apartado 21827, Caracas 1020A, Venezuela. Fax: 58-2-504-1479. E-mail: jaurbina{at}cbb.ivic.ve.

* Corresponding author. Mailing address for Wanderley de Souza: Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Bloco G, Cidade Universitária, 21949-900, Rio de Janeiro, Brazil. Fax: 55-21-260-2364. E-mail: wsouza{at}biof.ufrj.br.

Antimicrobial Agents and Chemotherapy, February 2002, p. 487-499, Vol. 46, No. 2
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.2.487-499.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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