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ABSTRACTAlthough the de novofolate biosynthesis pathway has been well studied in bacteria, little is known about its regulation. In the present study, the sigBgene in Mycobacterium tuberculosiswas deleted. Subsequent drug susceptibility tests revealed that the M. tuberculosisΔsigBstrain was more sensitive to para-aminosalicylic acid (PAS) and sulfamethoxazole. Comparative transcriptional analysis was performed, and downregulation of pabBwas observed in the ΔsigBstrain, which was further verified by a quantitative reverse transcription-PCR and Western blot assay. Then, the production levels of para-aminobenzoic acid (pABA) were compared between the sigBdeletion mutant and wild-type strain, and the results showed that sigBdeletion resulted in decreased production of pABA. In addition, SigB was able to recognize the promoter of pabB in vitro. Furthermore, we found that deleting pabCalso caused increased susceptibility to PAS. Taken together, our data revealed that, in M. tuberculosis, sigBaffects susceptibility to antifolates through multiple ways, primarily by regulating the expression of pabB. To our knowledge, this is the first report showing that SigB modulates pABA biosynthesis and thus affecting susceptibility to antifolates, which broadens our understanding of the regulation of bacterial folate metabolism and mechanisms of susceptibility to antifolates.

ABSTRACTCo-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 (pabBand nudB) involved in the folate biosynthesis of Escherichia coliwere deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of nudBresulted in a lag of growth in minimal medium and increased susceptibility to both SMX and TMP. Moreover, deletion of nudBalso greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of nudBaffects 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 nudBdeletion strain were isolated to further study the mechanism of how the deletion of nudBaffects 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 nudBdeletion. Meanwhile, overexpression of folMcould also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of nudBresulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of nudBalso resulted in increased susceptibility to both SMX and TMP in Salmonella enterica. 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.