Adriana Ángeles-Arvizu1, Sergio Enriquez-Flores2, Alma Jiménez-Gutiérrez1, Armando Pérez-Rangel3, Juan Pedro Luna-Arias3, Araceli Castillo-Romero4, José Manuel Hernández5, Gloria León-Avila6. 1. Departamento de Zoología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala S/N, Casco de Santo Tomás, Ciudad de México, 11340, México. 2. Grupo de Investigación en Biomoléculas y Salud Infantil, Laboratorio de EIMyT, Instituto Nacional de Pediatría, Ciudad de México, 04530, México. 3. Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN. Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, Ciudad de México, 07360, México. 4. Departamento de Microbiología y Patología, Centro Universitario de Ciencias de La Salud, Universidad de Guadalajara, Sierra Mojada 950, Col. Independencia, Guadalajara, 44340, México. 5. Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN. Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, Ciudad de México, 07360, México. manuel.hernandez@cinvestav.mx. 6. Departamento de Zoología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala S/N, Casco de Santo Tomás, Ciudad de México, 11340, México. leonavila60@yahoo.com.mx.
Abstract
BACKGROUND: Giardia intestinalis is a worldwide parasite. Drugs used for the treatment of giardiasis are metronidazole, albendazole and nitazoxanide. The development of drug resistance is an obstacle to the effective treatment. Resistance mechanisms in some parasites involve the participation of ATP-binding cassette (ABC) transporter superfamily. PURPOSE: To find if the ATP-binding cassette genes are overexpressed in trophozoites treated with albendazole or nitazoxanide. METHODS: A search for ATP-binding cassette genes in Giardia sequence database (GiardiaDB) was done and six genes were selected. Trophozoites treated with albendazole or nitazoxanide and the expression of these six ABC genes was quantitated by real-time RT-PCR. The ABC-C1 gene was selected, and a fragment cloned. The ABC-C1 protein was expressed, and polyclonal antibodies were elicited in mice to detect the protein in treated trophozoites, finally a docking analysis was performed for ABC-C1 and tizoxanide interaction. RESULTS: Bioinformatics analysis showed that the ATP-binding cassette (ABC) topology is present in the six proteins. The qRT-PCR revealed that the ABC-C1 gene was overexpressed in cells incubated with nitazoxanide or albendazole. Confocal analysis showed that ABC-C1 protein levels increased in trophozoites with both treatments but was higher with nitazoxanide. The mark was detected heavily in the periphery of the cells. Using a docking analysis, it was found that the nitazoxanide metabolite, tizoxanide was docked close to the ATP-binding region as well as in the exit tunnel, located in the transmembrane region. CONCLUSION: These findings in Giardia intestinalis, support the possible role of ABC-C1 in drug efflux.
BACKGROUND: Giardia intestinalis is a worldwide parasite. Drugs used for the treatment of giardiasis are metronidazole, albendazole and nitazoxanide. The development of drug resistance is an obstacle to the effective treatment. Resistance mechanisms in some parasites involve the participation of ATP-binding cassette (ABC) transporter superfamily. PURPOSE: To find if the ATP-binding cassette genes are overexpressed in trophozoites treated with albendazole or nitazoxanide. METHODS: A search for ATP-binding cassette genes in Giardia sequence database (GiardiaDB) was done and six genes were selected. Trophozoites treated with albendazole or nitazoxanide and the expression of these six ABC genes was quantitated by real-time RT-PCR. The ABC-C1 gene was selected, and a fragment cloned. The ABC-C1 protein was expressed, and polyclonal antibodies were elicited in mice to detect the protein in treated trophozoites, finally a docking analysis was performed for ABC-C1 and tizoxanide interaction. RESULTS: Bioinformatics analysis showed that the ATP-binding cassette (ABC) topology is present in the six proteins. The qRT-PCR revealed that the ABC-C1 gene was overexpressed in cells incubated with nitazoxanide or albendazole. Confocal analysis showed that ABC-C1 protein levels increased in trophozoites with both treatments but was higher with nitazoxanide. The mark was detected heavily in the periphery of the cells. Using a docking analysis, it was found that the nitazoxanide metabolite, tizoxanide was docked close to the ATP-binding region as well as in the exit tunnel, located in the transmembrane region. CONCLUSION: These findings in Giardia intestinalis, support the possible role of ABC-C1 in drug efflux.
Authors: Stephen G Aller; Jodie Yu; Andrew Ward; Yue Weng; Srinivas Chittaboina; Rupeng Zhuo; Patina M Harrell; Yenphuong T Trinh; Qinghai Zhang; Ina L Urbatsch; Geoffrey Chang Journal: Science Date: 2009-03-27 Impact factor: 47.728