Influence of dTMP on the phenotypic appearance and intracellular persistence of Staphylococcus aureus

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

^* To whom correspondence should be addressed. Email: Johannes_Zander{at}web.de.

	Abstract

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 in terms of small or normal colony variants (NCVs) is postulated to be affected by the intracellular amount of dTMP. This hypothesis was proven by metabolic pathway assays, leading to altered intracellular dTMP concentrations, followed by investigation of the associated phenotype. Inhibition of the staphylococcal thymidylate synthase, generating intracellular dTMP from dUMP, by 5-fluorouracil and cotrimoxazole, resulted in an SCV phenotype. Inhibition of a nucleoside transporter, providing the bacterial cell with extracellular thymidine, caused growth inhibition of SCVs. Reversion of SCVs to NCVs in turn was achieved by extracellular dTMP supply. 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 in cystic fibrosis cells revealed that clinical and cotrimoxazole-induced SCVs, with a diminished amount of dTMP, showed a significant better intracellular persistence compared to NCVs.

In conclusion, these results elucidate that the dTMP concentration plays a key role in both phenotypic appearance and intracellular persistence of S. aureus.