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

Abstract

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 0 -azido-3 0 -deoxythymidine (AZT) in the concentration range 0.032 -31.6 mM 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 K m of 73.3 mM and k cat /K m of 6.6 3 10 4 s 21 M 21 and is the activator of this drug in vivo. 2 0 ,2 0 -Difluoro-2 0 -deoxycytidine (gemcitabine) was a potent antibiotic against Gram-positive bacteria in the concentration range between 0.001 and 1.0 mM. The B. cereus deoxyadenosine kinase had a K m for gemcitabine of 33.5 mM and k cat /K m of 5.1 3 10 3 s 21 M 21 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.

Validation of a High-Throughput Screening Assay for Identification of Adjunctive and Directly Acting Antimicrobials Targeting Carbapenem-Resistant Enterobacteriaceae

“…44 Therefore, AZT also acts as an antibacterial agent, and previous studies have reported activity against Escherichia, Enterobacter, Klebsiella, Salmonella, and Shigella. [44][45][46][47] Our findings are therefore consistent with prior studies performed with a small number of carbapenem-susceptible Enterobacteriaceae strains. Notably, the antibiotics, trimethoprim and sulfamethoxazole, also interfere with DNA synthesis through modulation of the thymidine salvage pathway.…”

Adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase mutants combined with gemcitabine harbor a safe cancer treatment profile

“…Bcap37, SGC-7901, and MRC-5 cells were seeded in 6-well plates at a density of 5x10 5 cells/well and cultured for 24 h, followed by infection with ZD55-dNK and ZD55 at a MOI rate of 1. Two days later, cell protein extracts were prepared as described (20). The assays were performed in 50 mM of Tris-HCl, a pH of 7.6, 5 mM of MgCl 2 , 5 mM of ATP, 2 mM of dithiothreitol, 15 mM of NaF, 100 mM of KCl, 0.5 mg/ml bovine serum albumin, and 0.6 mg of protein extract in a total volume of 35 ml.…”

Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

“…Recombinant AtTK1a, AtTK1b and AtTK1b-dN45 were expressed and purified as described previously [34]. AtdNK was also purified in accordance with this procedure but with 2 mM MgCl 2 , 2 mM MnCl 2 and 2 mM ATP added to all buffers to stabilize the enzyme.…”