The ThiL enzyme is a valid antibacterial target essential for both thiamine biosynthesis and salvage pathways in .

Thiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. Thiamine biosynthesis occurs in bacteria but not in humans; therefore, the enzymes in this pathway are attractive targets for antibiotic development. Among these enzymes, thiamine monophosphate kinase (ThiL) catalyzes the final step of this pathway, phosphorylating thiamine monophosphate to produce TPP. Here, we extensively investigated ThiL in , a major pathogen responsible for hospital-acquired infections. We demonstrate that deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability and results in growth defects of the Δ strain even in the presence of thiamine derivatives, except for TPP. Most importantly, the pathogenesis of the Δ strain was markedly attenuated, compared with that of WT cells, with lower inflammatory cytokine induction and 10-10-fold decreased bacterial loads in an infection model in which the intracellular TPP level was in the submicromolar range. To validate ThiL (PaThiL) as a drug target, we further characterized its biochemical properties, determining a of 4.0 ± 0.2 nmol·min and values of 111 ± 8 and 8.0 ± 3.5 μm for ATP and thiamine monophosphate, respectively. An small-molecule screening assay identified PaThiL inhibitors including WAY213613, a noncompetitive inhibitor with a value of 13.4 ± 2.3 μm and potential antibacterial activity against These comprehensive biological and biochemical results indicate that PaThiL represents a potential drug target for the development of an augmented repertoire of antibiotics against .