Effect of Antibiotics and Antimicrobial Peptides on Single Protein Motility

Abstract

Following the movement of individual molecules of a bacterial surface protein in vivo we investigated the effects of antibiotics and antimicrobial peptides on protein motility and membrane structure. In previous work we engineered the lambda-receptor of Escherichia coli such that less than one receptor per cell is in vivo biotinylated and can bind to a streptavidin coated bead. Such a bead served as a handle for the optical tweezers to follow the motion of an individual receptor. In an un-perturbed living cell the lambda-receptor performs a confined diffusive motion. The lambda-receptor links to the peptidoglycan layer, and indeed, a perturbation of the peptidoglycan layer had a pronounced effect on the motility of the receptor: The motility significantly decreases upon treatment with vancomycin or ampicillin, to study the effect of vancomycin we used strains with increased membrane permeability. As the motility of an individual receptor was monitored over an extended amount of time we were able to observe a temporal evolution of the action of vancomycin. Antimicrobial peptides (AMPs) are alternatives to conventional antibiotics in the treatment of bacterial infections. Therefore, we also investigated the effect of the toxic AMP polymyxin B (PMB) which targets both the outer and inner membranes and kills the organism. PMB significantly decreased the motility of the lambda-receptor. On the basis of these findings we confirm that the lambda-receptor is firmly attached to the peptidoglycan layer, and that an antibiotic or AMP mediated destruction of the dynamic peptidoglycan synthesis decreases the receptor motion.

Manipulation and Motion of Organelles and Single Molecules in Living Cells

“…The motion of the lambda receptor was shown to be diffusive, with a larger diffusion constant than typically observed for protein motion in eukaryotic membranes, meanwhile having an elastic response due to cell wall tethering . Interestingly, the motion was shown to be strongly dependent on the bacterial metabolism − (i.e., the motion contained an active component which required a fully functional metabolism).…”

Optical manipulation of single molecules in the living cell

“…Also, the dynamics of the l-receptor was found to be energy dependent 81 and to correlate with the dynamic reassembly of the peptidoglycan layer. 82 These findings indicated that the l-receptor is attached to the peptidoglycan layer and that its diffusion is closely linked to the metabolism of the cell, hence it is not a pure thermal diffusion. These results suggest that membrane diffusion studied in vitro will be different from in vivo studies as the active component linked to cell metabolism is lacking in vitro.…”

Force probing of individual molecules inside the living cell is now a reality

“…4b), was shown to be connected more tightly to the membrane than typical eukaryotic membrane proteins and to have a higher diffusion constant 37,48 . Interestingly, the diffusion of the l-receptor is dependent on the bacterial metabolism of the host cell: if the cell is depleted of energy, the proteins move significantly less 14,49 . Figure 4c shows a time series of the positions visited by a particular l-receptor both before and after poisoning the cell with azide and arsenate to effectively stop electron transport and ATP synthesis.…”