Research Article: The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

Date Published: November 4, 2009

Publisher: Public Library of Science

Author(s): Mourad Ferhat, Danièle Atlan, Anne Vianney, Jean-Claude Lazzaroni, Patricia Doublet, Christophe Gilbert, Stefan Bereswill.

Abstract: Pneumonia associated with Iegionnaires’s disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds.

Partial Text: Legionella pneumophila (L. pneumophila), the main causative agent of the Legionnaire’s disease in humans is commonly found in freshwater environments where it can replicate within protozoa [1]. Development of air-conditioning systems, cooling towers and other water aerosols has created conditions for the direct access of this opportunistic bacterium to human lungs, where it can multiply within alveolar macrophages [2]. During the intracellular infectious cycle in amoeba and macrophages, L. pneumophila evades the phagolysosome fusion and establishes a replicative vacuole studded with ribosomes [1], [3], [4]. The subsequent modified vacuole offers a nutrient rich environment, which allows the bacteria to replicate. When nutrients become scarce, the bacteria undergo a phenotypic switch leading to the expression of virulent traits including flagella expression and an increased resistance to antibiotics and stress. Finally, Legionellae lyse the vacuole and the membrane host, probably by secreting pore-forming toxins [5].

Our analysis of the L. pneumophila Lens genome revealed a unique ORF sharing significant homology with the TolC protein from Escherichia coli, which is the prototypical outer membrane channel component involved in MDR and type I secretion [22], [23], [24]. The L. pneumophila Lens tolC::kan mutant was sensitive to a variety of compounds including antibiotics and detergents, which supports the involvement of the TolC protein in a functional MDR machinery with wide substrate specificity. The TolC pump activity was clearly demonstrated by the increase of ethidium bromide accumulation in the TolC deficient strain compared to the parental strain. The trans-complementation of tolC restored the pumping efficiency suggesting that the drug sensitivities were specifically due to the tolC knockout. Thus, our work confirms the role of L. pneumophila TolC in drug efflux mechanisms.

We thank Maëlle Molmeret and Christophe Ginevra (INSERM E230 – Faculté de médecine RTH Laennec 7–11 rue Guillaume Paradin 69372 Lyon Cedex 08) for the help with the macrophages infection experiments.



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