Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria

Abstract

Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against pathogenic staphylococci and streptococci. We show that pyrimidine-based nucleoside analogs, like 3'-azido-3'-deoxythymidine (AZT) and 2',2'-difluoro-2'deoxycytidine (gemcitabine), are specifically activated by the endogenous bacterial deoxyribonucleoside kinases, leading to cell death. Deoxyribonucleoside kinase-deficient Escherichia coli strains become highly susceptible to nucleoside analogs when they express recombinant kinases from Staphylococcus aureus or Streptococcus pyogenes. We further demonstrate that recombinant S. aureus deoxyadenosine kinase efficiently phosphorylates the anticancer drug gemcitabine in vitro and is therefore the key enzyme in the activation pathway. When adult mice were infected intraperitoneally with a fatal dose of S. pyogenes strain AP1 and afterwards received gemcitabine, they failed to develop a systemic infection. Nucleoside analogs may therefore represent a promising alternative for combating pathogenic bacteria.

FIG. 1.

Gemcitabine is an efficient antibacterial compound. A viable count antibacterial assay was performed for S. aureus ATCC 29213 (A) and S. pyogenes AP1 (B) in the stationary or exponential phase of growth. Gemcitabine effectively kills both bacteria when they are in exponential phase.

FIG. 2.

Phylogeny of the cloned dNKs. Three and two putative dNK genes were found in the S. aureus and S. pyogenes genomes, respectively. The encoded amino acid sequences were analyzed for their phylogenetic relationship with other dNKs. The phylogenetic relationships of TKs (A) and non-TKs (B) are shown. Both trees were reconstructed by the neighbor-joining method. The GenBank accession numbers are in brackets, and bootstrap values from 100 replications are given at the nodes. Apparently, S. aureus contains a gram-positive-type TK and two bacterial-type dAKs and dGKs, whereas S. pyogenes contains a gram-negative-type TK and one bacterial-type dAK.

FIG. 3.

Susceptibility to AZT and gemcitabine of E. coli KY895 transformed with S. pyogenes and S. aureus dNKs. (A) When transformed with SaTK and SpTK, KY895 shows impaired growth at 3.16 nM AZT and complete inhibition at 10.0 nM. AZT had no visible effect on the cells transformed with SadAK, SadGK, or SpdAK. (B) When transformed with SadAK, KY895 shows impaired growth at 0.316 nM gemcitabine and complete inhibition at 1.0 nM. When transformed with SpdAK, the cells could not grow at 31.6 nM gemcitabine. When cells were transformed with SpTK, SaTK, or SadGK, no inhibition was observed.

FIG. 4.

Kinetics of S. aureus recombinant dNKs, showing concentration-dependent reaction velocities for SaTK with AZT (A) and SadAK with gemcitabine (B). AZT confers characteristic substrate inhibition, resulting in declining reaction velocities at concentrations above 100 μM. In comparison, SadAK showed normal Michaelis-Menten kinetics with gemcitabine as the substrate. The figure clearly illustrates that dAK phosphorylates gemcitabine.

FIG. 5.

Gemcitabine protects animals from bacterial dissemination during systemic infection. BALB/c female mice were subjected to a systemic infection with S. pyogenes AP1. Six hours after initiation of infection, the animals were treated with either gemcitabine or PBS (control), and 24 h after initiation of infection, the bacterial load in the spleen was determined for each animal. Each dot represents an individual animal.

FIG. 6.

Kaplan-Meier survival curve for BALB/c mice after intraperitoneal infection with S. pyogenes AP1. Female BALB/c mice were subjected to a systemic infection with S. pyogenes AP1. Six hours after initiation of infection, the animals were treated with either gemcitabine or PBS (control), and the survival within the group was monitored. All six (100%) animals in the untreated group died of streptococcal infection within 30 h of infection. Only one of six (17%) animals treated with gemcitabine died as a result of streptococcal infection.