Abstract
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 Pseudomonas aeruginosa, a major pathogen responsible for hospital-acquired infections. We demonstrate that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability and results in growth defects of the ΔthiL strain even in the presence of thiamine derivatives, except for TPP. Most importantly, the pathogenesis of the ΔthiL strain was markedly attenuated, compared with that of WT cells, with lower inflammatory cytokine induction and 103-104-fold decreased bacterial loads in an in vivo infection model in which the intracellular TPP level was in the submicromolar range. To validate P. aeruginosa ThiL (PaThiL) as a drug target, we further characterized its biochemical properties, determining a Vmax of 4.0 ± 0.2 nmol·min-1 and Km values of 111 ± 8 and 8.0 ± 3.5 μm for ATP and thiamine monophosphate, respectively. An in vitro small-molecule screening assay identified PaThiL inhibitors including WAY213613, a noncompetitive inhibitor with a Ki value of 13.4 ± 2.3 μm and potential antibacterial activity against P. aeruginosa These comprehensive biological and biochemical results indicate that PaThiL represents a potential drug target for the development of an augmented repertoire of antibiotics against P. aeruginosa.
Highlights
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen associated with a wide range of acute and chronic infections of various body sites, including the urinary tract, skin, and respiratory tract [1]
Complementation with the plasmid expressing thiL relieved the growth defect of the DthiL mutant caused by thiamine pyrophosphate (TPP) depletion, suggesting that thiamine monophosphate kinase (ThiL) is essential for TPP biosynthesis i0n P. aeruginosa.sss
Two types of direct thiamine salvage pathways have been identified in bacteria, namely, one-step pyrophosphorylation of thiamine to TPP by thiamine pyrophosphokinase (TPK/ThiN) and two steps of subsequent monophosphorylation of thiamine to thiamine monophosphate (TMP) and TPP by thiamine kinase (ThiK) and ThiL, respectively
Summary
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen associated with a wide range of acute and chronic infections of various body sites, including the urinary tract, skin, and respiratory tract [1]. Complementation with the plasmid expressing thiL (pthiL) relieved the growth defect of the DthiL mutant caused by TPP depletion, suggesting that ThiL is essential for TPP biosynthesis i0n P. aeruginosa (see Fig. 2A).sss
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