Deletion of Causes Increased Susceptibility to Antifolates in Escherichia coli and Salmonella enterica.

Co-trimoxazole, a fixed-dose combination of sulfamethoxazole (SMX) and trimethoprim (TMP), has been used for the treatment of bacterial infections since the 1960s. Since it has long been assumed that the synergistic effects between SMX and TMP are the consequence of targeting 2 different enzymes of bacterial folate biosynthesis, 2 genes ( and ) involved in the folate biosynthesis of were deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of resulted in a lag of growth in minimal medium and increased susceptibility to both SMX and TMP. Moreover, deletion of also greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of affects the bacterial growth and susceptibility to antifolates, 7,8-dihydroneopterin and 7,8-dihydropteroate were supplemented into the growth medium. Although those metabolites could restore bacterial growth, they had no effect on susceptibilities to the antifolates. Reverse mutants of the deletion strain were isolated to further study the mechanism of how the deletion of affects susceptibility to antifolates. Targeted sequencing and subsequent genetic studies revealed that the disruption of the tetrahydromonapterin biosynthesis pathway could reverse the phenotype caused by the deletion. Meanwhile, overexpression of could also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of resulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of also resulted in increased susceptibility to both SMX and TMP in Since dihydroneopterin triphosphate hydrolase is an important component of bacterial folate biosynthesis and the tetrahydromonapterin biosynthesis pathway also exists in a variety of bacteria, it will be interesting to design new compounds targeting dihydroneopterin triphosphate hydrolase, which may inhibit bacterial growth and simultaneously potentiate the antimicrobial activities of antifolates targeting other components of folate biosynthesis.