title: A covalent reporter of β-lactamase activity for fluorescent imaging and rapid screening of antibiotic-resistant bacteria.
abstract: Bacterial resistance to antibiotics poses a great clinical challenge in fighting serious infectious diseases due to complicated resistant mechanisms and time-consuming testing methods. Chemical reaction-directed covalent labeling of resistance-associated bacterial proteins in the context of a complicated environment offers great opportunity for the in-depth understanding of the biological basis conferring drug resistance, and for the development of effective diagnostic approaches. In the present study, three fluorogenic reagents LRBL1-3 for resistant bacteria labeling have been designed and prepared on the basis of fluorescence resonance energy transfer (FRET). The hydrolyzed probes could act as reactive electrophiles to attach the enzyme, β-lactamase, and thus facilitated the covalent labeling of drug resistant bacterial strains. SDS electrophoresis and MALDI-TOF mass spectrometry characterization confirmed that these probes were sensitive and specific to β-lactamase and could therefore serve for covalent and localized fluorescence labeling of the enzyme structure. Moreover, this β-lactamase-induced covalent labeling provides quantitative analysis of the resistant bacterial population (down to 5%) by high resolution flow cytometry, and allows single-cell detection and direct observation of bacterial enzyme activity in resistant pathogenic species. This approach offers great promise for clinical investigations and microbiological research.
authors.affiliation: Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore.
authors.forename: Qing
authors.initials: Q
authors.lastname: Shao
authors.forename: Yan
authors.initials: Y
authors.lastname: Zheng
authors.forename: Xueming
authors.initials: X
authors.lastname: Dong
authors.forename: Kai
authors.initials: K
authors.lastname: Tang
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Yan
authors.forename: Bengang
authors.initials: B
authors.lastname: Xing
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001419:Bacteria / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D001618:beta-Lactamases
keywords: FRET; cell cytometry; covalent labeling; fluorescent probes; β-lactamase