Deciphering the mode of action of the synthetic antimicrobial peptide Bac8c

Abstract

Bac8c (RIWVIWRR-NH(2)) is an 8-amino-acid peptide derived from Bac2A (RLARIVVIRVAR-NH(2)), a C3A/C11A variant of the naturally occurring bovine peptide, bactenecin (also known as bovine dodecapeptide), the smallest peptide with activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. The effects of Bac8c on Escherichia coli were examined by studying its bacteriostatic and bactericidal properties, demonstrating its effects on proton motive force generation, and visually analyzing (via transmission electron microscopy) its effects on cells at different concentrations, in order to probe the complexities of the mechanism of action of Bac8c. Results were consistent with a two-stage model for the Bac8c mode of action. At sublethal concentrations (3 μg/ml), Bac8c addition resulted in transient membrane destabilization and metabolic imbalances, which appeared to be linked to inhibition of respiratory function. Although sublethal concentrations resulted in deleterious downstream events, such as methylglyoxal formation and free radical generation, native E. coli defense systems were sufficient for full recovery within 2 h. In contrast, at the minimal bactericidal concentration (6 μg/ml), Bac8c substantially but incompletely depolarized the cytoplasmic membrane within 5 min and disrupted electron transport, which in turn resulted in partial membrane permeabilization and cell death.

FIG. 1.

Effects of Bac8c on growth and survival of E. coli cells. (A) Effects of Bac8c on the specific growth rate of E. coli (Mach-1 T1; Invitrogen). Bac8c was added to exponentially growing cultures (time zero [x axis], OD₆₀₀ of 0.1), and the growth rate was determined at intervals of 15 to 30 min for 4 h after Bac8c addition. (B) Effects of Bac8c exposure on survival of exponentially growing E. coli cells. Bac8c was added at time zero, and cells were monitored over 3 h (OD₆₀₀, 0.1).

FIG. 2.

Membrane permeabilization (A and C) and depolarization (B and D). We examined the role of membrane permeability by using the membrane-impermeable dye TO-PRO-3. (A) Bac8c at an inhibitory concentration (3 μg/ml) did not cause an increase in membrane permeability over time. (C) After 15 min, Bac8c at the MBC caused a 1-log increase in membrane permeability in approximately 14.5% of the population, while after 60 min 65% of the population was permeabilized (see Table 1). (B and D) In parallel, membrane depolarization over time was examined in E. coli by using the dye DiSCO(3)₂. (B) At the Bac8c IC₅₀, membrane depolarization occurred within 5 min, and the population recovered from the insult within 90 min. (D) Membrane depolarization occurred within 5 min at the Bac8c MBC, and over time depolarization increased to 64% after 30 min. After 60 min of exposure, depolarization increased to 74% (data not shown). OD₆₀₀, 0.1.

FIG. 3.

Transmission electron microscopy findings. Logarithmically growing E. coli cells were cultured in MOPS minimal medium in the presence or absence of various concentrations of Bac8c. Samples were taken after 30 min, and cells were fixed in glutaraldehyde immediately and then processed for TEM. (A) Control; (B) 3 μg/ml (IC₅₀); (C) 6 μg/ml (MBC); (D) 30 μg/ml (5× the MBC). (E to G) At a higher magnification, the control (E), cells exposed to 6 μg/ml Bac8c (F), and cells exposed to 30 μg/ml Bac8c (G) are shown. (H to K) Alternate images of cells at the MBC (6 μg/ml), focused on the membrane effects of Bac8c.

FIG. 4.

Macromolecular synthesis. Incorporation of ³H-labeled radioactive isotopes was measured over time. (A) The CFU/ml was sampled at the same time isotope incorporation was measured. (B) Thymidine incorporation was measured to study the effects of Bac8c on DNA replication. Below the MBC, incorporation was unaffected. (C) Uridine incorporation was measured to study the effects of Bac8con transcription. Effects on transcription were seen at 40 μg/ml. (D) Histidine incorporation was measured to study the effects of Bac8c on translation. All concentrations of Bac8c had some effect on translation. In panels B to D, the y axis represents counts per minute (CPM) of radioactivity. Symbols: squares, control; diamonds, 2 μg/ml Bac8c; triangles, 4 μg/ml Bac8c; crosses, 40 μg/ml Bac8c (all in MOPS minimal medium). Cultures were started at an OD₆₀₀ of 0.2.

FIG. 5.

Bac8c inhibition of ATP synthesis and electron transport chain activity. (A) ATP concentration in logarithmically grown E. coli cells under normal conditions (diamonds), at the inhibitory concentration (crosses; 3 μg/ml), and the MBC (triangles; 6 μg/ml). (B) E. coli NAD⁺/NADH ratios after exposure to Bac8c (6 μg/ml) decreased as a function of time, reflecting decreased redox or electron transport. (C) The decrease in redox was a function of an increase in NADH and was concentration dependent. (D) Conditions that stimulated NAD⁺ biosynthesis also showed an increase in sublethal resistance to Bac8c. We tested the effects of supplementation with the NAD⁺ precursors nicotinamide (NAD⁺ salvage pathway), aspartate (NAD⁺ biosynthesis I), and glutamine (NAD⁺ biosynthesis I, glutamine dependent).

FIG. 6.

Methylglyoxal accumulation and hydroxyl radical formation. (A) Intracellular accumulation of MG occurs after 2 h of incubation with sublethal levels of Bac8c (sub-MIC level of Bac8c or 6 μg/ml; OD, 0.5). (B) Flow cytometry measurement of the increase in hydroxyl radical formation by the dye HPF after 2 h with control (filled light gray), cells exposed to the IC₅₀ (3 μg/ml; dark gray), and cells exposed to the MBC (6 μg/ml; black). Hydroxyl radical formation after 60 min of exposure to Bac8c was not significantly different from the control. OD₆₀₀, 0.1.

FIG. 7.

Bac8c MOA timeline. After addition of Bac8c, the first two events that occur are a decrease in the redox potential, due to an increase in NADH, and partial membrane depolarization. Within 15 min, ATP is depleted, and inhibition of macromolecular synthesis occurs. At this time, over 50% of cells have completely depolarized, yet only 15% of this population is permeabilized. Within 30 min, there is less than 10% cell survival, and there is a corresponding increase in membrane depolarization and permeabilization. Within 60 min, most of the population observed is depolarized and permeated, and 99% of the population is nonrecoverable. After 60 min, the accumulation of both MG and free radicals can be observed.