Elucidating Duramycin's Bacterial Selectivity and Mode of Action on the Bacterial Cell Envelope

Abstract

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical, and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and Populus-derived bacterial isolates to determine species selectivity. Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE) in the cell membrane. Further the surface and interface morphology were studied by high resolution atomic force microscopy and showed a progression of cellular changes in the cell envelope after treatment with duramycin for the susceptible bacterial strains. Together, these molecular and cellular level analyses provide insight into duramycin's mode of action and a better understanding of its selectivity.

Keywords: atomic force microscopy (AFM); cell elasticity; duramycin; lipid; lipidomics; molecular adhesion force; peptidoglycan; phosphatidylethanolamine (PE).

FIGURE 1

Growth curves of bacterial strains treated with duramycin. The growth curves of Arthrobacter CF158 and Pseudomonas GM17, the two resistant bacterial strains and Bacillus subtilis 168 and Bacillus BC15, the two sensitive strains, in the presence of various concentrations of duramycin in R2A growth media are shown. In the absence of duramycin, no major differences in growth rates are observed between the strains. Further, for the resistant strains, Arthrobacter CF158 and Pseudomonas GM17, duramycin concentrations up to 2 μM do not affect bacterial growth. However, growth of Bacillus BC15 and B. subtilis 168 is inhibited at 500 and 200 nM duramycin, respectively. Figures show the average data of three biological measurements, and standard deviations were all less than 3%.

FIGURE 2

Growth curve comparisons for first and second-generation cells exposed to duramycin. (A) First-generation bacteria grown without and with 1 μM duramycin. (B) Second-generation bacteria grown without and with 1 μM duramycin. Growth of the second-generation Bacillus BC15 and B. subtilis 168 strains, in presence of 1 μM duramycin, are improved relative to the first-generation cells. Figures show the average data of three biological measurements, and standard deviations were all less than 3%.

FIGURE 3

Lipodomic analyses of duramycin sensitive and resistant strains. (A) The phospholipid species profiles are shown for the indicated duramycin resistant (top) and sensitive cells (bottom). The sensitive, Gram positive strains contain high levels of PE. (B) Shows the phospholipid species profiles for the same cells that have been conditioned with duramycin. All cell strains have changed their phospholipid profiles. PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; CL, cardiolipin.

FIGURE 4

Time course AFM images of duramycin resistant and sensitive cells. (A) The duramycin resistant strains, Arthrobacter CF158 and Pseudomonas GM17, show no change in surface ultrastructure and morphology with increasing duramycin exposure time while the duramycin sensitive strains, Bacillus BC15 and B. subtilis 168, show progressive cell destruction. Images were collected at time zero, before duramycin treatment, and after 1–3 h of duramycin treatment. (B) The morphology changes between first-generation and duramycin conditioned, second-generation cells, without duramycin treatment, can be compared. The previously sensitive Bacillus BC15 and B. subtilis 168 strains show altered morphology, especially the B. subtilis 168 strain.

FIGURE 5

Molecular recognition experiments using duramycin functionalized AFM tips. AFM tips were functionalized with duramycin for interacting with PE on the cell membrane. A graph of the adhesion force frequency versus rupture strength was generated by a 16 × 16 point scan of a 0.5 μm × 0.5 μm sized area on the top of the bacterial cell. An average of 10 different cells from Arthrobacter CF158, Pseudomonas GM17, Bacillus BC15, and B. subtilis 168 were taken. Force curves collected on Pseudomonas GM17, Bacillus BC15, and B. subtilis 168 indicate a high-frequency of strong adhesion between the cell surface and the tip with an adhesion force of ∼3 × 10^-9 N for the three bacteria. There is also a small amount of adhesion force measured for Arthrobacter CF158. In order to show that the interaction of duramycin on the tip is specific for PE, free PE was added to the imaging solution and then the adhesion force was measured using Bacillus BC15. This treatment blocks the duramycin functionalized tip and prevents adhesion to PE on the cell surface.