Genome-scale metabolic modelling of trimethoprim's mode of action reveals free ATP salvaged to restore purine pool

Trimethoprim is a clinically important antibiotic for treating urinary tract infections yet its mechanism of killing remains elusive due to a cascade of effected metabolic reactions. Metabolites available in growth media can selectively counteract trimethoprim which affects the interpretation of results. We sought to understand the full scope of trimethoprims impact on Escherichia coli metabolism and how metabolite availability affects trimethoprim outcomes. We applied flux balance analysis on a genome-scale metabolic model of E. coli to simulate trimethoprim activity under bacteriostatic and bactericidal conditions. Our results suggested that in the absence of environmental purines or nucleosides, trimethoprim induces salvage of ATP to repair DNA. We experimentally validated the result with ATP bioluminescence screening of 96 clinical E. coli isolates. Therefore, the choice of growth media composition significantly changes the outcome of trimethoprim challenge and should be taken into consideration when interpreting trimethoprims activity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/666800v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@cddc46org.highwire.dtl.DTLVardef@138a743org.highwire.dtl.DTLVardef@6d4857org.highwire.dtl.DTLVardef@a8502d_HPS_FORMAT_FIGEXP M_FIG C_FIG Diagrammatic representation of trimethoprim action on susceptible UPEC in bacteriostatic and bactericidal conditions. The diagram flows downwards in a time-irrespective manner with outcomes on cellular functions/properties not in order of events. The half-tone at the end represents slowing down of cellular process, however, the outcome is not clear in bactericidal conditions. Circular, green, glowing element at the end of ATP is a representation of ATP bioluminescence. Further, thinning of the line for viability and ATP denotes a reduction in the respective property. RNA and DNA components are labelled above the trimethoprim line with solid grey-to-yellow lines denoting components unaffected by trimethoprim activity. The components that are affected by trimethoprim are replenished by ATP, adenine, or uracil (U) and are denoted by the flow of lines. Abbreviations: A, adenine; C, cytosine; G, guanine; T, thymidine; U, uracil. Sources: Kwon et al. 1, Amyes and Smith 2, Makino and Munakata 6, this study.