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Influence of dTMP on the Phenotypic Appearance and Intracellular Persistence of Staphylococcus aureus

Johannes Zander,^1*, Silke Besier,^1, Stephan H. Saum,² Faramarz Dehghani,³ Stefan Loitsch,⁴ Volker Brade,¹ and Thomas A. Wichelhaus¹

Institute of Medical Microbiology and Infection Control, Hospital of Johann Wolfgang Goethe University, 60596 Frankfurt/Main, Germany,¹ Institute of Molecular Biosciences, University, Frankfurt/Main, Germany,² Institute of Anatomy II, University Hospital, Frankfurt/Main, Germany,³ Department of Medicine II, University Hospital, Frankfurt/Main, Germany⁴

Received 3 August 2007/ Returned for modification 24 September 2007/ Accepted 14 December 2007

Thymidine-dependent small-colony variants (SCVs) of Staphylococcus aureus are frequently associated with persistent and recurrent infections in cystic fibrosis patients. The phenotypic appearance of S. aureus SCVs or normal-colony variants (NCVs) is postulated to be affected by the intracellular amount of dTMP. This hypothesis was proven by metabolic pathway assays revealing altered intracellular dTMP concentrations, followed by investigation of the associated phenotype. Inhibition of the staphylococcal thymidylate synthase, which generated intracellular dTMP from dUMP, using 5-fluorouracil and co-trimoxazole resulted in an SCV phenotype. Inhibition of a nucleoside transporter, which provided the bacterial cell with extracellular thymidine, caused growth inhibition of SCVs. In turn, reversion of SCVs to NCVs was achieved by supplying extracellular dTMP. High-performance liquid chromatography additionally confirmed the intracellular lack of dTMP in SCVs, in contrast to NCVs. Moreover, the dTMP concentration is postulated to influence the intracellular persistence of S. aureus. Cell culture experiments with cystic fibrosis cells revealed that clinical and co-trimoxazole-induced SCVs with a diminished amount of dTMP showed significantly better intracellular persistence than NCVs. In conclusion, these results show that the dTMP concentration plays a key role in both the phenotypic appearance and the intracellular persistence of S. aureus.

Published ahead of print on 26 December 2007.

Editor: B. A. McCormick

J.Z. and S.B. contributed equally to this work.