title: A Novel Metallo-β-Lactamase Involved in the Ampicillin Resistance of Streptococcus pneumoniae ATCC 49136 Strain.
abstract: Streptococcus pneumoniae, a penicillin-sensitive bacterium, is recognized as a major cause of pneumonia and is treated clinically with penicillin-based antibiotics. The rapid increase in resistance to penicillin and other antibiotics affects 450 million people globally and results in 4 million deaths every year. To unveil the mechanism of resistance of S. pneumoniae is thus an important issue to treat streptococcal disease that might consequently save millions of lives around the world. In this work, we isolated a streptococci-conserved L-ascorbate 6-phosphate lactonase, from S. pneumoniae ATCC 49136. This protein reveals a metallo-β-lactamase activity in vitro, which is able to deactivate an ampicillin-based antibiotic by hydrolyzing the amide bond of the β-lactam ring. The Michaelis parameter (Km) = 25 μM and turnover number (kcat) = 2 s(-1) were obtained when nitrocefin was utilized as an optically measurable substrate. Through confocal images and western blot analyses with a specific antibody, the indigenous protein was recognized in S. pneumoniae ATCC 49136. The protein-overexpressed S. pneumonia exhibits a high ampicillin-tolerance ability in vivo. In contrast, the protein-knockout S. pneumonia reveals the ampicillin-sensitive feature relative to the wild type strain. Based on these results, we propose that this protein is a membrane-associated metallo-β-lactamase (MBL) involved in the antibiotic-resistant property of S. pneumoniae.
authors.affiliation: Department of Applied Chemistry, National Chiao Tung University, 1001 University Road, Hsinchu, Taiwan.
authors.forename: Chia-Yu
authors.initials: CY
authors.lastname: Chang
authors.affiliation: Department of Applied Chemistry, National Chiao Tung University, 1001 University Road, Hsinchu, Taiwan.
authors.forename: Hui-Jen
authors.initials: HJ
authors.lastname: Lin
authors.affiliation: Institute of Biomedical Engineering, National Chiao Tung University, 1001 University Road, Hsinchu, Taiwan.|Center for Interdisciplinary Science, National Chiao Tung University, 1001 University Road, Hsinchu, Taiwan.
authors.forename: Bor-Ran
authors.identifier: 0000-0003-2266-680X
authors.initials: BR
authors.lastname: Li
authors.affiliation: Department of Applied Chemistry, National Chiao Tung University, 1001 University Road, Hsinchu, Taiwan.|Center for Interdisciplinary Science, National Chiao Tung University, 1001 University Road, Hsinchu, Taiwan.
authors.forename: Yaw-Kuen
authors.initials: YK
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000668:Ampicillin Resistance; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D055786:Gene Knockout Techniques; D006868:Hydrolysis; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; D001618:beta-Lactamases