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Antimicrobial Agents and Chemotherapy, June 2009, p. 2410-2416, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01428-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of the Efflux Transporter of the Fluoroquinolone Antibiotic Ciprofloxacin in Murine Macrophages: Studies with Ciprofloxacin-Resistant Cells{triangledown}

Béatrice Marquez, Nancy E. Caceres,{dagger} Marie-Paule Mingeot-Leclercq, Paul M. Tulkens, and Françoise Van Bambeke*

Unité de Pharmacologie Cellulaire et Moléculaire, Louvain Drug Research Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium

Received 23 October 2008/ Returned for modification 3 March 2009/ Accepted 16 March 2009

Ciprofloxacin, the most widely used totally synthetic antibiotic, is subject to active efflux mediated by a MRP-like transporter in wild-type murine J774 macrophages. To identify the transporter among the seven potential Mrps, we used cells made resistant to ciprofloxacin obtained by long-term exposure to increasing drug concentrations (these cells show less ciprofloxacin accumulation and provide a protected niche for ciprofloxacin-sensitive intracellular Listeria monocytogenes). In the present paper, we first show that ciprofloxacin-resistant cells display a faster efflux of ciprofloxacin which is inhibited by gemfibrozil (an unspecific MRP inhibitor). Elacridar, at a concentration known to inhibit P-glycoprotein and breast cancer resistance protein (BCRP), only slightly increased ciprofloxacin accumulation, with no difference between resistant and wild-type cells. Analysis at the mRNA (real-time PCR) and protein (Western blotting) levels revealed an overexpression of Mrp2 and Mrp4. Mrp4 transcripts, however, were overwhelmingly predominant (45% [wild-type cells] to 95% [ciprofloxacin-resistant cells] of all Mrp transcripts tested [Mrp1 to Mrp7]). Silencing of Mrp2 and Mrp4 with specific small interfering RNAs showed that only Mrp4 is involved in ciprofloxacin transport in both ciprofloxacin-resistant and wild-type cells. The study therefore identifies Mrp4 as the most likely transporter of ciprofloxacin in murine macrophages but leaves open a possible common upregulation mechanism for both Mrp4 and Mrp2 upon chronic exposure of eukaryotic cells to this widely used antibiotic.

* Corresponding author. Mailing address: Unité de Pharmacologie Cellulaire et Moléculaire, UCL 7370 Avenue Mounier 73, B-1200 Brussels, Belgium. Phone: 32-2-764-73-78. Fax: 32-2-764-73-73. E-mail: francoise.vanbambeke{at}uclouvain.be

{triangledown} Published ahead of print on 23 March 2009.

{dagger} Present address: Ludwig Institute for Cancer Research, Signal Transduction Unit, B-1200 Brussels, Belgium.

Antimicrobial Agents and Chemotherapy, June 2009, p. 2410-2416, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01428-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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