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JAC Advance Access originally published online on July 5, 2007
Journal of Antimicrobial Chemotherapy 2007 60(3):510-520; doi:10.1093/jac/dkm240
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner

Michael P. B. Sandrini1,2,*,{dagger}, Anders R. Clausen2, Stephen L. W. On3,{ddagger}, Frank M. Aarestrup3, Birgitte Munch-Petersen4 and Jure Piskur2

1 BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark 2 Cell and Organism Biology, Lund University, Sölvegatan 35, SE-22362 Lund, Sweden 3 Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen, Denmark 4 Department of Science, Systems and Models, Roskilde University, DK-4000 Roskilde, Denmark

Received 26 March 2007; returned 27 April 2007; revised 5 June 2007; accepted 8 June 2007

* Corresponding author. Tel: +45-3532-1734; E-mail: msandrini{at}aki.ku.dk

Objectives: To investigate the bactericidal activity of antiviral and anticancer nucleoside analogues against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs).

Methods: Several FDA-approved nucleoside analogue drugs were screened for their potential bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes. These genes were tested for their ability to increase the susceptibility of a dNK-deficient E. coli strain to various analogues. We overexpressed, purified and characterized the substrate specificity and kinetic properties of the recombinant enzymes from S. enterica and B. cereus.

Results: The tested Gram-negative bacteria were susceptible to 3'-azido-3'-deoxythymidine (AZT) in the concentration range 0.032–31.6 µM except for a single E. coli isolate and two Pseudomonas aeruginosa isolates which were resistant to the tested AZT concentrations. Purified recombinant S. enterica thymidine kinase phosphorylated AZT efficiently with a Km of 73.3 µM and kcat/Km of 6.6 x 104 s–1 M–1 and is the activator of this drug in vivo. 2',2'-Difluoro-2'-deoxycytidine (gemcitabine) was a potent antibiotic against Gram-positive bacteria in the concentration range between 0.001 and 1.0 µM. The B. cereus deoxyadenosine kinase had a Km for gemcitabine of 33.5 µM and kcat/Km of 5.1 x 103 s–1 M–1 and activates gemcitabine in vivo. S. enterica and B. cereus are now amongst the first bacteria with a completely characterized set of dNK enzymes.

Conclusions: Bacterial dNKs efficiently activate nucleoside analogues in a species-specific manner. Therefore, nucleoside analogues have a potential to be employed as antibiotics in the fight against emerging multiresistant bacteria.

Keywords: antibacterial , multidrug-resistant , thymidine kinase , salvage

{dagger} Present address. Department of Molecular Biology, Copenhagen University, Copenhagen, Denmark.

{ddagger} Present address. Institute of Environmental Science and Research, Christchurch, New Zealand.


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