Abstract
Multidrug efflux pumps play an essential role in antibiotic resistance. The conventional methods, including minimum inhibitory concentration and fluorescent assays, to monitor transporter efflux activity might have some drawbacks, such as indirect evidence or interference from color molecules. In this study, MALDI-TOF MS use was explored for monitoring drug efflux by a multidrug transporter, and the results were compared for validation with the data from conventional methods. Minimum inhibitory concentration was used first to evaluate the activity of Escherichia coli drug transporter AcrB, and this analysis showed that the E. coli overexpressing AcrB exhibited elevated resistance to various antibiotics and dyes. Fluorescence-based studies indicated that AcrB in E. coli could decrease the accumulation of intracellular dyes and display various efflux rate constants for different dyes, suggesting AcrB’s efflux activity. The MALDI-TOF MS analysis parameters were optimized to maintain a detection accuracy for AcrB’s substrates; furthermore, the MS data showed that E. coli overexpressing AcrB led to increased ions abundancy of various dyes and drugs in the extracellular space at different rates over time, illustrating continuous substrate efflux by AcrB. This study concluded that MALDI-TOF MS is a reliable method that can rapidly determine the drug pump efflux activity for various substrates.
Highlights
Multidrug transporters are cell membrane glycoproteins that excrete various classes of compounds across cell membranes
The migration of the AcrB protein, which has 1049 amino acid residues with a predicted molecular mass of approximately 113 kDa, was between 93 and 125 kDa (Supplementary Figure S1A, lane E)
Further identification of AcrB was accomplished using LC-MS/MS, with 41% sequence coverage (Supplementary Figure S1B). These data indicated that AcrB protein was successfully overexpressed in E. coli Kam3
Summary
Multidrug transporters are cell membrane glycoproteins that excrete various classes of compounds across cell membranes. Multidrug transporters are classified into two categories: ATP binding cassette transporters and secondary active transporters. The drug excretion systems that utilize the free energy from ATP hydrolysis to actively transport substrates across a membrane belong to the ATP binding cassette transporters group [1,2]. On the other hand, are driven by a proton motive force. These transporters have been categorized into several families in terms of their protein sequence similarities: the major facilitator superfamily (MFS), the multidrug and toxic compound extrusion (MATE) family, the small multidrug resistance (SMR) family and the resistance nodulation division (RND) family [3,4]. One of the most studied secondary active transporters is Antibiotics 2020, 9, 639; doi:10.3390/antibiotics9100639 www.mdpi.com/journal/antibiotics
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