Effect of Thymidine on the Activity of Diaminopyrimidine Antibacterial Agents:

1. Effect of Thymidine on the Activity of Diaminopyrimidine Antibacterial Agents: Generation and Characterization of Thymidine Kinase-Deficient Staphylococcus aureus Mutants A. HALDIMANN, S. HAWSER, M. BIHR, S. FELS, S. RIGO, L. WEISS, K. ISLAM, S. LOCIURO; ARPIDA AG, Muenchenstein, Switzerland 1.00E+11 1.00E+09 Figure 3. Growth of S. aureus Strains 1.00E+07 Table 3. Effect of Thymidine on Iclaprim MBC CFU / mL 1.00E+05 MICMBC Strain - dT + dT (1 mg/mL) - dT + dT (1 mg/mL) ATCC 25923 0.06 0.06 0.06 32 AH1246 (tdk-) 0.06 0.06 0.060.06 ATCC 25923 100 10 g/mL] 1 m MIC [ 0.1 0.01 0 2 4 6 8 10 wild-type mutant thymidine [mg/mL] Figure 7. Effect of Thymidine on Iclaprim Killing a) ATCC 25923 (Wild-Type) Growth control Growth control 10 4x MIC iclaprim 4x MIC iclaprim 9 4x MIC iclaprim + 1 mg/mL dT 4x MIC iclaprim + 1 mg/mL dT 8 4x MIC iclaprim + 10 mg/mL dT 4x MIC iclaprim + 10 mg/mL dT 7 4x MIC vancomycin 4x MIC vancomycin 6 4x MIC vancomycin + 1 mg/mL dT 4x MIC vancomycin + 1 mg/mL dT CFU / mL (log10) 4x MIC vancomycin + 10 mg/mL dT 4x MIC vancomycin + 10 mg/mL dT 5 4 3 0 0 5 5 10 10 15 15 20 20 25 25 2 1 wild-type mutant trimethoprim iclaprim co-trimoxazole Time (hours) b) AH1246 (TK Mutant) 10 9 8 7 6 Figure 4. Thymidine Incorporation CFU / mL (log10) 5 4 3 2 1 Time (hours) wild-type mutant Figure 8. Effect of Thymidine on Iclaprim PAE a) ATCC 25923 (Wild-Type) Table 1. Plasma Thymidine Levels Figure 1. Biochemical Pathways Species Thymidine conc. Human 2.5 ± 0.2 ng/mL Mouse 822 ± 63 ng/mL Rat 267 ± 9 ng/mL GTP MH MH MH + 1 µg/mL dT MH + 1 µg/mL dT 2.5 Figure 5. Nucleoside Uptake (10 min reaction) 2 1.5 PAE (hours) 1000 1 0.5 Table 2. Trimethoprim MICs DHPS DHPS AH1249 Smx Smx ATCC 25923 ATCC 25923 0 L613 L613 3817 3817 AW6 AW6 AH1246 AH1252 - thymidine + thymidine (50 mg/mL) ATCC 25923 1 >32 24 variants 0.25-0.5 0.25-1 AW6 0.25 >32 24 variants 0.125-0.50.125-0.5 L613 64 >512 24 variants 16-64 32-128 3817 1 >32 24 variants 0.125-0.5 0.125-1 DHFR DHFR AH1255 THF THF Iclaprim 1x MIC Iclaprim 2x MIC Iclaprim 4x MIC Iclaprim 8x MIC Iclaprim 0.5x MIC 100 DHF DHF relative CPM (%) AH1252 wild-type mutant Tmp Tmp AH1255 AH1246 mTHF mTHF AH1249 thymine (FUdR) dCMP dCMP b) AH1246 (TK Mutant) 10 dUMP dUMP dTMP dTMP thymidine thymidine TS TS TK TK dUDP dUDP Smx 1 fluoro-uracil de de novo novo salvage salvage thymidine uridine 2.5 Tmp 2 DNA DNA Figure 6. Changes Found in Mutant TK Genes 1.5 PAE (hours) 1 IS IS X X 0.5 nt nt 0 C183Y C183Y Q137 V113D V113D M17K M17K Q137 + 1 + 1 Iclaprim 2x MIC Iclaprim 4x MIC Iclaprim 8x MIC Iclaprim 1x MIC Iclaprim 0.5x MIC N N - - - - C C Lasso region / Zn++ motifs (dT binding) P-loop (ATP binding) C1-940 Contact Information Andreas Haldimann Arpida Ltd Dammstrasse 36 4142 Muenchenstein SWITZERLAND Phone: +41 61 417 9671 Fax: +41 61 417 9661 E-mail: ahaldimann@arpida.ch Methods Bacterial growth Cation-adjusted Mueller-Hinton media from Oxoid (Basel, Switzerland) were used throughout the study. These media were found to contain only minimal amounts of thymidine (data not shown). Growth of S. aureus strains was followed in an automated microtiter format using a Bioscreen C reader (Catalys AG, Wallisellen, Switzerland). Antimicrobial activities MIC (Minimal Inhibitory Concentration), MBC (Minimal Bactericidal Concentration) and time-kill experiments were performed under standard CLSI conditions. PAE (Post-antibiotic effect) studies were performed as described elsewhere (Jacobs et al., 2003). Genetic selection and analysis of TK mutants Mutations at the tdk locus in S. aureus were searched for essentially as described before (Summers and Raskin, 1993) by selecting resistance to 5-fluoro-deoxyuridine (FUdR; 10 mg/mL). tdk genes were PCR amplified using ProofStart high-fidelity DNA polymerase (Qiagen, Hombrechtikon, Switzerland), and cloned into a plasmid vector using standard procedures. DNA sequence determinations were done at Microsynth (Balgach, Switzerland). Nucleoside incorporation and uptake Incorporation and uptake of nucleosides were measured using a microtiter plate format. Essentially, S. aureus cells grown to exponential phase in diluted (25 %) Mueller-Hinton broth, were exposed for up to 40 minutes to [3H]thymidine or [3H]uridine. For incorporation studies cells were subsequently lysed using trichloroacetic acid (TCA) and precipitated on ice for 30 minutes. Whole cells (uptake) or TCA precipitates (incorporation) were then applied to MHABN45 filter plates (Millipore, Volketswil Switzerland), and washed on the filter. Finally, after addition of scintillation fluid, retained radioactivity was determined using a Packard TopCount scintillation counter (Perkin-Elmer, Schwerzenbach, Switzerland). Abstract Background: Trimethoprim, iclaprim, and other diaminopyrimidines (DAPs) specifically and selectively inhibit microbial dihydrofolate reductase and thus de novo biosynthesis of thymidine monophosphate (dTMP). Uptake of thymidine (dT) and its conversion into dTMP by thymidine kinase (TK) is a well known bypass mechanism leading to an increase in MIC of DAPs. E. coli mutants lacking the dTMP salvage pathway have been reported. Analogous S. aureus mutants are so far not known perhaps due to concerns about essentiality of tdk (encoding TK). In humans dT levels are extremely low (< 0.01 μg/mL) whereas in rodents dT levels as high as 1 μg/mL are reported. The confounding effects of dT in assessing potency of DAPs in animal infection models is well documented. Methods:S. aureus mutants were isolated by a genetic selection based on resistance to the dT analogue fluorodeoxyuridine (FUdR). tdk genes were sequenced. MICs, MBCs, time-kill kinetics and PAE were performed under CLSI guidelines where available. Results: Starting from 4 parent strains FUdRr mutants have been isolated and all showed the predicted marked decrease in dT incorporation. Sequence analysis of the respective tdk genes revealed various harmful mutations. Subsequently a deletion of the entire tdk gene was constructed and tested against standard folate pathway inhibitors. Addition of concentrations of dT such as those found in rodents (1 µg/mL) in the growth medium of S. aureus did not produce significant shifts in MICs, but did cause a significant loss in MBC of DAPs. In addition, the bactericidal activity of iclaprim (99.9 % killing at 5 hours) was lost by addition of 1 µg/mL and the PAE (0.8 – 2.3 hours) reduced by 50 %. When the effect of dT was studied on TK-deficient S. aureus mutants bactericidal activity and PAE of iclaprim were no longer affected. These mutants could represent a new tool for the study of the in vivo properties of DAPs against S. aureus. Figure 2. Influence of Thymidine on Activity of DAPs Introduction Trimethoprim, iclaprim, and other diaminopyrimidines (DAPs) specifically and selectively inhibit microbial dihydrofolate reductase and thus de novo biosynthesis of thymidine monophosphate (dTMP) (Figure 1). Uptake of thymidine (dT) and its conversion into dTMP by thymidine kinase (TK) is a well known bypass mechanism present in various bacteria (Hamilton-Miller, 1988; Tokunaga at al., 1997). Consequently, when dT is readily available in the bacterial growth environment dTMP de novo biosynthesis becomes more and more dispensable and renders the activity of e.g. trimethoprim significantly weaker. Importantly, dT levels in many animal species, especially rodents, are very high (Table 1; Li et al., 2003), and maybe even higher in infected animals (Nottebrock and Then, 1977). Such high levels are known to dramatically effect the efficacy of such molecules in vivo. On the contrary, dT levels in humans are extremely low (at least 200-fold lower than in rodents; Table 1) and do not affect the activity of DAPs in the human setting. In order to fully evaluate the efficacy of DAPs in animal models of infection, animals would be required to be essentially free of dT. As rodents can not be rendered dT free, we have genetically selected and characterized dT-independent S. aureus TK mutants and speculate on their utility for novel efficacy studies with DAP antibiotics.  • Results • Table 2 & Figure 2: S. aureus mutants were selected for which the DAPs MIC levels are unaffected by elevated thymidine levels. • Figure 3: In vitro these mutants grew as well as their wild-type parent strains. • Figure 4 & Figure 5: Mutants showed a severe defect in thymidine incorporation and uptake. This defect is limited to thymidine since the uptake of uridine is not compromised. • Figure 6: tdk – the gene encoding thymidine kinase – of these mutants contains mutations predicted to be critical for the enzymatic function. Subsequently, we succeeded in deleting tdk in strain ATCC 25923 (data not shown). Furthermore, in contrast to purified wild-type TKs, no enzymatic activity was identified with purified mutant TKs (data not shown). • Table 3: Relatively low thymidine levels (1 mg/mL) had no effect on the MIC but significantly attenuated the MBC of iclaprim in wild-type S. aureus. On the contrary, thymidine had no effect on the MIC or MBC regarding TK mutants. • Figure 7: In the absence of added thymidine, iclaprim exhibited a rapid bactericidal activity against both wild-type and TK mutants. However, against wild-type S. aureus iclaprim became at best bacteriostatic when tested in the presence of increasing concentrations of thymidine. On the contrary, iclaprim remained rapidly bactericidal against the TK mutant irrespective of the additions of high concentrations of thymidine. • Figure 8: In the absence of added thymidine, iclaprim exhibited a significant PAE against both wild-type and TK mutants. However, against wild-type S. aureus, iclaprim lost 50 % of its PAE when tested in the presence of thymidine. On the contrary, iclaprim retained its PAE on TK mutants irrespective of the addition of thymidine. • Conclusions • Selected mutants were shown to possess critical mutations in thymidine kinase, and were severely impaired in thymidine incorporation. • Mutants, unlike wild-type strains, were unaffected by addition of exogenous thymidine •  bactericidal activities of iclaprim, and iclaprim PAE were unaffected by thymidine. • The mutants may be considered as „humanized“ strains, and pending virulence in vivo are likely to be of important value in efficacy studies in animals. • References • Hamilton-Miller, J.M.T. (1988). J. Antimicrob. Chemother. 22:35-39. • Jacobs, M.R., S. Bajaksouzian, and P.C. Appelbaum (2003). J. Antimicrob. Chemother. 52: 809-812. • Li, K.M., S.J. Clarke, and L.P. Rivory (2003). Anal. Chim. Acta 486:51-61. • Nottebrock, H., and R. Then (1977). Biochem. Pharmacol. 26:2175-79. • Summers, W.C., and P. Raskin (1993). J. Bacteriol. 175:6049-51. • Tokunaga, T., K. Oka, A. Takemoto, Y. Ohtsubo, N. Gotoh, and T. Nishino (1997). Antimicrob. Agents Chemother. 41:1042-45.