Quantitation of Polymyxin-Lipopolysaccharide Interactions Using an Image-Based Fluorescent Probe

Abstract

The frequency of polymyxin-resistant pathogenic Gram-negative bacteria appearing in the clinic is increasing, and the consequences are largely mediated by modification of lipopolysaccharide (LPS) in the outer membrane. As polymyxins exert their antibacterial effect by binding to LPS, understanding their mode of binding will prove highly valuable for new antibiotic discovery. In this study, we assess the potential of MIPS-9451, a fluorescent polymyxin analogue designed for imaging studies, as a fluorescent reporter molecule, titrating it against 17 different Gram-negative species and/or strains of LPS. MIPS-9451 bound to the various species and/or strains of LPS with a dissociation constant ranging between 0.14 ± 0.01 μM (Escherichia coli) and 0.90 ± 0.42 μM (Porphyromonas gingivalis; mean ± standard error). Furthermore, we assessed the applicability of MIPS-9451 to rank affinities of polymyxin B to different LPS species in a displacement assay which yielded inhibition constants of 6.2 μM ± 33%, 7.2 μM ± 30%, and 0.95 μM ± 13% for Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella enterica, respectively (mean ± coefficient of variation). The results from this study are concordant with those observed with similarly structured polymyxin probes, confirming the potential of MIPS-9451 for quantitation of polymyxin-LPS affinities in discovery programs of novel polymyxin antibiotics.

Keywords: anti-infectives; drug resistance; fluorescence spectroscopy; in vitro models; peptides; structure–activity relationship.

Figure 1

A) Representative emission spectra illustrating fluorescence enhancement upon the titration of increasing MIPS-9451 concentration into S. enterica (typhimurium) LPS, where the upward arrow indicates a rise in the curve upon successive titrations and B) area under curve of each titration plotted as a function of MIPS-9451 concentration. Data are presented as mean ± SD (n = 3) and are fitted to a Hill-slope augmented one-site specific binding model.

Figure 2

A) Representative emission spectra illustrating fluorescence attenuation upon titration of increasing polymyxin B concentration against S. enterica LPS with the direction of the arrow indicating increasing polymyxin B concentration; and B) area under the curve of each titration plotted as a function of polymyxin B concentration. Data are presented as mean ± SD (n = 3) and fit to a competitive one-site binding model. Note: some data points were associated with very small error, thus the error bars are not visible.