Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner.
OBJECTIVE
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 microM 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 microM and k(cat)/Km of 6.6 x 10(4) 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 microM. The B. cereus deoxyadenosine kinase had a Km for gemcitabine of 33.5 microM and k(cat)/Km of 5.1 x 10(3) 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.
Sandrini
Michael P B
MP
BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark. msandrini@aki.ku.dk
Clausen
Anders R
AR
On
Stephen L W
SL
Aarestrup
Frank M
FM
Munch-Petersen
Birgitte
B
Piskur
Jure
J
D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects* / Q000201:enzymology*; D001711:Biotransformation / Q000187:drug effects; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009705:Nucleosides / Q000378:metabolism* / Q000494:pharmacology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity; D013937:Thymidine Kinase / Q000378:metabolism; D015215:Zidovudine / Q000494:pharmacology; D000093542:Gemcitabine
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D009705:Nucleosides; D011994:Recombinant Proteins; D003841:Deoxycytidine; D015215:Zidovudine; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D013937:Thymidine Kinase; D000093542:Gemcitabine