Bactericidal activity of the human skin fatty acid cis-6-hexadecanoic acid on Staphylococcus aureus

Abstract

Human skin fatty acids are a potent aspect of our innate defenses, giving surface protection against potentially invasive organisms. They provide an important parameter in determining the ecology of the skin microflora, and alterations can lead to increased colonization by pathogens such as Staphylococcus aureus. Harnessing skin fatty acids may also give a new avenue of exploration in the generation of control measures against drug-resistant organisms. Despite their importance, the mechanism(s) whereby skin fatty acids kill bacteria has remained largely elusive. Here, we describe an analysis of the bactericidal effects of the major human skin fatty acid cis-6-hexadecenoic acid (C6H) on the human commensal and pathogen S. aureus. Several C6H concentration-dependent mechanisms were found. At high concentrations, C6H swiftly kills cells associated with a general loss of membrane integrity. However, C6H still kills at lower concentrations, acting through disruption of the proton motive force, an increase in membrane fluidity, and its effects on electron transfer. The design of analogues with altered bactericidal effects has begun to determine the structural constraints on activity and paves the way for the rational design of new antistaphylococcal agents.

FIG 1

Characterization of the bactericidal effect of C6H. Effect of external pH on bactericidal activity. S. aureus cells were incubated (for 2 h at 37°C) in various buffers (20 mM N-cyclohexyl-3-aminopropanesulfonic acid [CAPS; pH 10], 25 mM HEPES [pH 7.4], 20 mM PBS [pH 7.4], 20 mM sodium citrate buffer [pH 5.5], and 20 mM MES [pH 5.5]). (A) Percentage of surviving cells with no C6H compared to the percentage of cells at 0 min. (B) Percent survival of the cells after incubation with 5 μg/ml C6H compared to percent survival of the cells with no C6H at the same time point. (C) Survival kinetics and the effect of KCl on C6H activity. The effect of 3 and 5 μg/ml C6H (● and ■, respectively) with (open symbols) or without (filled symbols) 50 mM KCl (▼, 10 μg/ml C6H). For all data points, the percent survival was compared to that of the wild type at the same time.

FIG 2

Effect of C6H on membrane integrity (A and B) and potential (C). (A) Cells were incubated with PI for 1 min and 20 mM MES before addition (black arrow) of 3 μg/ml C6H (black curve) and 5 μg/ml C6H (gray curve). (B) Effect of C6H on the rate of PI fluorescence increase. (C) Membrane potential was measured for 10 min before (at 0 min) the addition of 3 μg/ml C6H (■), 20 μg/ml valinomycin (▲), 3 μg/ml C6H, or 20 μg/ml valinomycin (▼) and after no addition (●).

FIG 3

C6H reduces the ability to control internal pH in S. aureus. Effect of C6H on fluorescence ratio of cFDASE (left ordinate) and corresponding pH (right ordinate). C6H (3 μg/ml) was added (filled arrow), followed by excess amounts of nigericin and valinomycin (white arrow).

FIG 4

Effect of C6H and various inhibitors on the electron transport chain of E. coli inverted vesicles. NADH (left) and succinate (right) were used as the substrates to generate the electron flow. Data are expressed as percent deviations in formazan production from substrates alone using the INT reduction assay.

FIG 5

C6H increases S. aureus membrane fluidity. Polarization index (PI) dynamics were measured after treatment of S. aureus cells with C6H or CCCP (0.5 μg/ml).

FIG 6

S. aureus morphology in the presence of C6H. Transmission electron microscopy was used to investigate the morphology of S. aureus after a 2-h standard killing assay in the presence (B) or absence (A) of 5 μg/ml C6H. Scale bars, 0.5 μm. The arrows highlight aberrant septation events.

FIG 7

Bactericidal activity of C6H analogues. (A) Various analogues of C6H of the structure shown were chemically synthesized and purified. (B) Antibacterial activity of C6H (●), C8 (▲), and a fluorescent derivative, Bodipy 1 (■). For all data points, the percent survival was compared to that of the wild type at the same time. All compounds were at 17 μM (equivalent to 5 μg/ml C8). ■, Bodipy; ●, C6H; ▲, C8. (C) Structure of Bodipy 1.

FIG 8

Subcellular localization of Bodipy 1. S. aureus SH1000 was treated with FL-vancomycin and Bodipy 1, washed, and visualized by fluorescence microscopy. (A) DIC; (B) Bodipy 1 labeling; (C) FL-vancomycin labeling; (D) overlay of panels B and C. (E) Line scan through the cell confirming the overall staining of Bodipy 1 labeling throughout the cell membrane.