ThiL is a valid antibacterial target that is essential for both thiamine biosynthesis and salvage pathway inPseudomonas aeruginosa

Abstract

ABSTRACTThiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. As thiamine biosynthesis occurs in bacteria but not humans, bacterial thiamine biosynthesis is an attractive target for antibiotic development. Among enzymes in the thiamine biosynthetic pathway, thiamine monophosphate kinase (ThiL) catalyzes the final step of the pathway, phosphorylating thiamine monophosphate (TMP) to produce TPP. In this work, we extensively investigated ThiL inPseudomonas aeruginosa, a major pathogen of hospital-acquired infections. We demonstrated thatthiLdeletion abolishes not only thiamine biosynthesis but also thiamine salvage capability, showing growth defects of the ΔthiLmutant even in the presence of thiamine derivatives except TPP. Most importantly, the pathogenesis of the ΔthiLmutant was markedly attenuated compared to wild-type bacteria, with lower inflammatory cytokine induction and 103~104times decreased bacterial load in anin vivoinfection model where the intracellular TPP level is in the submicromolar range. In order to validateP. aeruginosaThiL (PaThiL) as a new drug target, we further characterized its biochemical properties determining a Vmax of 4.0±0.2 nomol·min−1and KMvalues of 111±8 and 8.0±3.5μM for ATP and TMP, respectively. A subsequentin vitrosmall molecule screening identified PaThiL inhibitors including WAY213613 that is a noncompetitive inhibitor with a Ki value of 13.4±2.3 μM and a potential antibacterial activity againstP. aeruginosa. This study proved that PaThiL is a new drug target againstP. aeruginosaproviding comprehensive biological and biochemical data that could facilitate to develop a new repertoire of antibiotics.