Lipidated Short Analogue of α-Melanocyte Stimulating Hormone Exerts Bactericidal Activity against the Stationary Phase of Methicillin-Resistant Staphylococcus aureus and Inhibits Biofilm Formation.

Stationary phase Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has been widely associated with many persistent infections as well as biofilm-associated infections, which are challenging due to their increasing antibiotic resistance. α-Melanocyte stimulating hormone (α-MSH) is an antimicrobial peptide (AMP) with well-established potent activity against S. aureus , but little is known about its antimicrobial efficacy against the stationary phase of the bacteria. We investigated the in vitro activities of two palmitoylated analogues, Pal-α-MSH(6-13) and Pal-α-MSH(11-13), of the C-terminal fragments of α-MSH against biofilm-producing strains of methicillin-sensitive S. aureus (MSSA) and MRSA. While both the peptides demonstrated anti-staphylococcal efficacy, Pal-α-MSH(11-13) emerged as the most effective AMP as palmitoylation led to a remarkable enhancement in its activity against stationary phase bacteria. Similar to α-MSH, both the designed analogues were membrane-active and exhibited improved bacterial membrane depolarization and permeabilization, as further confirmed via electron microscopy studies. Of the two peptides, Pal-α-MSH(11-13) was able to retain its activity in the presence of standard microbiological media, which otherwise is a major limiting factor toward the therapeutic use of α-MSH-based peptides. More importantly, Pal-α-MSH(11-13) was also highly effective in inhibiting the formation of biofilms. Furthermore, it did not lead to resistance development in MRSA cells even upon 18 serial passages at sub-MIC concentrations. These observations support the potential use of Pal-α-MSH(11-13) in the treatment of planktonic as well as sessile S. aureus infections.

Introduction

Staphylococcus aureus, an opportunistic pathogen, is capable of acquiring antibiotic resistance traits with an ease that led to the rapid and global dissemination of the recalcitrant pathogen methicillin-resistant S. aureus (MRSA). Although MRSA is present asymptomatically in approximately 30% of the human population,[1] it harbours a wide variety of virulence determinants that enable this common colonizer of the human skin and nares to cause a range of diseases even in immunocompetent hosts. Most antibiotics depend upon the bacteria dividing actively to exhibit potent killing efficacy. In the case of many infections, such as endocarditis, prosthetic joint infections, and infected embedded catheters, the bacteria divide slowly or remain dormant, i.e., do not divide at all. Stationary phase bacteria are a cause of several systemic infections including biofilm-associated infections and are often refractory to antibiotic treatment as they are slow-growing and exhibit a metabolically quiescent state.[2,3] Biofilms are bacterial communities with an extremely high number of bacterial cells embedded in a self-produced polymeric matrix.[4] Along with presenting a physical barrier for antibiotic penetration and protection from host immune surveillance, biofilms resist antibiotics by modification at the genetic level in microbes.[2] However, these are not the only reasons for the chronicity and recalcitrance of biofilm infections. The unique environmental milieu and high cell density present in biofilms lead to conditions of nutrient and oxygen limitation that cause a subset of the bacterial cells embedded in the biofilms to exist in the stationary phase, and as such, biofilms may display high levels of multidrug tolerance.[5,6] Clinically used antibiotics, although highly potent against planktonic cells, rarely retain bactericidal activities against nondividing or stationary phase cells.[7−10] Thus, compounds with effective killing mechanisms against the stationary phase and biofilm of S. aureus cells may serve as new therapeutic agents.

Cationic antimicrobial peptides (CAMPs) are gene-encoded short stretches of amino acid residues (12–60 amino acids) with cationic and hydrophobic amino acids, which present an amphipathic arrangement upon membrane interaction.[11] CAMPs and their mimics are lucrative alternatives to antibiotics as they can target multiple critical cellular functions in microbes, including membrane disruption, which is believed to overcome the drug resistance problem.[12,13] However, the therapeutic applications of CAMPs have been impeded by several issues, such as low stability, toxicity, and a high cost of production.[14] In order to overcome these issues, there is a lot of interest in the development of novel CAMPs with modifications that render them more suitable for therapeutic applications. Toward this, lipopeptides have emerged as a promising class of CAMP mimics with direct bactericidal activity as well as potential to reinvigorate conventional antibiotics by synergy and immune modulation.[15−18] A number of clinically approved cyclic lipopeptides such as daptomycin, teicoplanin, and polymyxins constitute another class of antibiotics, which are currently of paramount clinical significance.[15] According to a study by Yarlagadda et al., the attachment of lipids to glycopeptide vancomycin increased its activity against vancomycin-resistant enterococci by 300-fold.[19] Another study showed that a short antibacterial (RW)3 sequence that was only active against Gram-positive bacteria exhibited a substantial increase in its activity against both Gram-positive and Gram-negative bacteria (including Pseudomonas aeruginosa and Acinetobacter baumannii) upon attachment of a side-chain lipidated lysine residue at its N- or C-terminal.[20]

Our group has been perusing the host neuropeptide α-melanocyte stimulating hormone (α-MSH) as a lead molecule against S. aureus.[21−25] α-MSH is a short, linear, endogenous tridecapeptide derived from pro-opiomelanocortin with primary sequence Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2. With +1 unit charge and a turn conformation in the membrane mimic environment, it has been shown to have potent antipyretic, anti-inflammatory, and pigmentary effects.[26] We have already established potent and rapid antibacterial activity of α-MSH and its shorter analogues (α-MSH(6-13) and α-MSH(11-13)) against S. aureus, MRSA, and multiple clinical isolates of S. aureus in a low micromolar concentration range.[21,22] Interestingly, the C-terminal fragment α-MSH(11-13) has been shown to have potent antibacterial activity upon covalent conjugation with different chain length fatty acids.[27] Further, recently, we have reported that enhanced cationic charge significantly improved the staphylocidal potential of α-MSH-based analogues by promoting the interaction of the cationic peptides with anionic lipid membranes.[28] However, a major limitation to the therapeutic potential of α-MSH-based peptides is their diminished antimicrobial activity in the presence of standard bacterial growth media.[22,23,25,28,29]

Thus, in this study, we aimed to develop lipidated analogues of the C-terminal fragments of α-MSH, i.e., α-MSH(6-13) and α-MSH(11-13), which would not only be able to overcome the barrier of α-MSH inactivity in complex biological growth media but also be efficacious against the stationary phase and biofilm of S. aureus, which are clinically more relevant. First, we studied the activity of the designed analogues in the presence of buffer against the stationary phase of methicillin-sensitive S. aureus (MSSA) as well as MRSA. Next, we evaluated the secondary structure of the analogues, as well as their interaction with artificial bacterial membrane mimics, through CD spectroscopy and Trp fluorescence studies, respectively. We then determined the toxicity of the designed analogues against mammalian cells. The mechanism of action of the analogues was delineated using depolarization as well as permeabilization assays of the bacterial membrane, and it was further corroborated via electron microscopy studies. We also assessed the ability of the analogues to retain their staphylocidal potential in the presence of bacterial growth media, and the active analogue was further studied to determine whether it could inhibit the formation of bacterial biofilm. Finally, the ability of MRSA cells to develop resistance against the active analogue was also examined via a serial passage study.

Results

Antimicrobial Activity of the Analogues against the Stationary Phase of S. aureus

In this study, we designed two N-terminal lipidated analogues, i.e., palmitoylated α-MSH(6-13) (Pal-α-MSH(6-13)) and palmitoylated α-MSH(11-13) (Pal-α-MSH(11-13)), as shown in Table .

Table 1

Name, Sequence, Molecular Mass, and Charge of the Palmitoylated Analogues and their Parent Peptides

name	sequence	molecular mass (Da)	charge
α-MSH	Ac-S1-Y2-S3-M4-E5-H6-F7-R8-W9-G10-K11-P12-V13-NH2	1664.88	+1
α-MSH(6-13)	Ac-H6-F7-R8-W9-G10-K11-P12-V13-NH2	1067.25	+2
α-MSH(11-13)	Ac-K11-P12-V13-NH2	383.49	+1
Pal-α-MSH(6-13)	palmitoyl-H6-F7-R8-W9-G10-K11-P12-V13-NH2	1263.77	+2
Pal-α-MSH(11-13)	palmitoyl-K11-P12-V13-NH2	579.9	+1

Initially, we determined the killing efficacy of the C-terminal fragments of α-MSH, i.e., α-MSH(6-13) and α-MSH(11-13), against 105 CFU/mL stationary phase cells of S. aureus in 10 mM phosphate-buffered saline (PBS; 150 mM NaCl, pH 7.4). As seen in Figure a, α-MSH(6-13) caused ∼2 log reduction in the bacterial cells after 2 h incubation at 20 and 50 μM concentrations, while α-MSH(11-13) even at 150 μM concentration could reduce the viable cell count by only 0.7 log after 2 h incubation (Figure b).

Figure 1

Antibacterial activity of (a) α-MSH(6-13) and (b) α-MSH(11-13) against 105 CFU/mL MSSA ATCC 29213. The bacterial cells were incubated with different concentrations of the peptides in PBS for 2 h. Each data point represents mean ± SEM. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Upon palmitoylation of α-MSH(6-13), it was seen that Pal-α-MSH(6-13) showed activity similar to that of α-MSH(6-13) against the stationary phase of S. aureus, exhibiting approximately 1 and 2 log reductions in viable cell count upon 1 and 2 h incubation, respectively, at 20 μM concentration (Figure a). However, there was a drastic improvement in the activity of α-MSH(11-13) against stationary phase cells upon palmitoylation (Figure b). Within 30 min of incubation with Pal-α-MSH(11-13) at only 8.6 μM concentration, the viable cell count of MSSA was reduced by 1 log (Figure b,i). This reduction in viability at 8.6 μM increased to 1.5 and 2 log at the 1 and 2 h time points, respectively. Upon increasing the concentration to 17.2 μM, within 30 min, there was a 2.7 log reduction in the viable cell count, and the peptide showed a bactericidal effect reducing the MSSA cells by 3.8 log at the 1 h time point. Here, the bactericidal effect is defined as a >3 log reduction in viability in comparison to the untreated control. This effect was also exhibited by 34.4 μM Pal-α-MSH(11-13) within only 30 min of incubation (4.5 log reduction), and there was complete eradication of 105 CFU/mL MSSA cells within 1 h. Furthermore, Pal-α-MSH(11-13) demonstrated similar potency against MRSA cells, as seen in Figure b,ii and it also completely eradicated 105 CFU/mL MRSA cells within 1 h of incubation with 34.4 μM concentration of the peptide.

Figure 2

(a) Antibacterial activity of Pal-α-MSH(6-13) against MSSA ATCC 29213. (b) Antibacterial activity of Pal-α-MSH(11-13) against (i) MSSA ATCC 29213 and (ii) against MRSA ATCC 33591. The bacterial cells were incubated with different concentrations of the peptides in PBS for 2 h. Each data point represents mean ± SEM. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Thus, out of the two designed palmitoylated peptides, Pal-α-MSH(11-13) showed excellent and rapid bactericidal activity against S. aureus cells under physiological buffer conditions.

Secondary Structure of the Peptides in Different Environments and their Interaction with Model Membranes by CD Spectroscopy and Tryptophan Fluorescence

To understand the difference in the effect that palmitoylation had upon the staphylocidal potential of α-MSH(6-13) and α-MSH(11-13), we examined their oligomeric state and structure (if any) when bound to bacterial membrane mimic small unilamellar vesicles (SUVs). Because Pal-α-MSH(11-13) is a short 3-mer peptide and lacks a tryptophan moiety, these experiments could only be performed for α-MSH(6-13) and Pal-α-MSH(6-13).

The secondary structure of α-MSH(6-13) and Pal-α-MSH(6-13) was measured in different environments, viz., 5 mM phosphate buffer (PB), 50% (v/v) TFE, and DMPC/DMPG (7:3, w/w) bacterial membrane mimic SUVs, via CD spectroscopy (Figure ). Surprisingly, while the buffer spectrum of α-MSH(6-13) was that of a typical peptide in a random conformation, with a negative extremum at 201 nm and a small positive peak at 226 nm, Pal-α-MSH(6-13), in buffer, exhibited a maximum at 200 nm and a minimum at 220 nm. This suggests that Pal-α-MSH(6-13) had a distinct secondary structure even in the presence of buffer alone. There was no substantial difference in the secondary structures in the presence of 50% (v/v) TFE and DMPC/DMPG (7:3 w/w) bacterial mimic SUVs upon palmitoylation of α-MSH(6-13).

Figure 3

Circular dichroism spectra of (a) α-MSH(6-13) and (b) Pal-α-MSH(6-13) in 5 mM PB (blue), 50% TFE (green), and DMPC/DMPG (7:3, w/w) SUVs (pink). Lipid concentration was 1453 μM and peptide concentration was 35 μM (lipid/peptide ratio of 41.5:1). The spectrum of each peptide was the average of two scans and was plotted as ellipticity [θ] against wavelength (nm).

Similarly, the Trp emission study revealed that the Trp moiety of Pal-α-MSH(6-13) was already in a hydrophobic environment even in the presence of only buffer as its emission maximum, i.e., 337 nm, showed a blue shift as compared to that of α-MSH(6-13), i.e., 352 nm. Typically, when a peptide interacts with the vesicles such that there is an alteration in the microenvironment of its Trp residue, a blue shift and variation in quantum yield are expected.[30] Here, in the absence of any such environments, our designed analogue Pal-α-MSH(6-13) exhibited a blue shift, suggesting a defined structure in the presence of buffer itself (Figure ).

Figure 4

Trp fluorescence emission spectra of (a) α-MSH(6-13) and (b) Pal-α-MSH(6-13) in buffer (blue), DMPC (green), and DMPC/DMPG (7:3, w/w) (pink) SUVs. Lipid concentration was 726 μM and peptide concentration was 14.5 μM (lipid/peptide ratio of 50:1).

Toxicity Studies of the Designed Peptides toward Mammalian Cells

As an initial characterization of the selective potential of the designed peptides, we studied their hemolytic effect on murine red blood cells (RBCs) and their cytotoxicity against 3T3 murine fibroblast cells. For the hemolysis study, we used 0.1% Triton X-100, a non-ionic surfactant, as the positive control (100% hemolysis) and determined the HC50 value (concentration that causes lysis of 50% of the RBCs) of the designed peptides. As seen in Figure a, Pal-α-MSH(6-13) showed negligible hemolysis (≤1%) up to the tested concentration of 62.5 μM, similar to the parent peptide. For Pal-α-MSH(11-13), the HC50 value was determined to be 62.5 μM, which is more than the concentration required to exhibit bactericidal effect against both stationary phase MSSA and MRSA cells. At the same time, melittin, a naturally occurring AMP found in bee venom, known to show strong hemolytic activity due to its poor cell selectivity,[31] exhibited an HC50 value of ≤3.9 μM.

Figure 5

(a) Percentage hemolysis of murine RBCs upon 1 h treatment with the palmitoylated peptides and their parent peptides. 0.1% Triton X-100 was used as a positive control (100% lysis) for the experiment. (b) Cytotoxicity of palmitoylated peptides toward the 3T3 murine fibroblast cell line. The cytotoxicity of the peptides was evaluated as percentage viability of the murine cells upon treatment with palmitoylated peptides for 2 h relative to untreated growth control. The experiments were performed in duplicate on two different days. Each data point represents mean ± SEM.

For the cytotoxicity experiment, we incubated 3T3 murine fibroblast cells with two different concentrations of the palmitoylated peptides, viz., 20 and 50 μM. In Figure b, Pal-α-MSH(6-13) did not exhibit any detrimental effect on the viability of the fibroblast cells even at 50 μM and even the most active peptide, Pal-α-MSH(11-13), at 20 and 50 μM, resulted in ≥85.5% viable cells. 2% Triton X-100, used as a positive control, showed a 52.8% reduction in survival of the cells relative to untreated growth control. Of note in this experiment, we did not supplement the DMEM with FBS during incubation of the cells with peptides as it can potentially lead to false negatives.[32]

Thus, the palmitoylated peptides showed little to no cytotoxicity toward mammalian cells at the tested concentrations.

Mechanism of Action of the Designed Peptides against Stationary Phase S. aureus Strains

To explore whether the designed peptides could be targeting the S. aureus membrane as previously reported for their parent peptides,[22] we carried out experiments with DiSC3(5), a potentiometric probe, and determined the ability of Pal-α-MSH(6-13) and Pal-α-MSH(11-13) to depolarize the stationary phase staphylococcal membrane.

Under the experimental conditions, in both MSSA (Figure a) and MRSA (Figure b) cells resuspended in 5 mM HEPES (20 mM glucose, pH 7.2) buffer, the C-terminal fragments of α-MSH caused only a marginal increase in the fluorescence intensity of DiSC3(5), up to the maximum concentration tested, i.e., 40 μM. However, upon palmitoylation, both α-MSH(6-13) and α-MSH(11-13) peptides were able to induce instant depolarization of the bacterial membrane in a concentration-dependent manner as an increase in fluorescence was seen within ∼2 min of peptide addition. Pal-α-MSH(11-13) showed higher membrane depolarizing capability than Pal-α-MSH(6-13) against both stationary phase MSSA (Figure a) and MRSA cells (Figure b). In parallel, we studied the corresponding viability of the dye-loaded cells at 20 μM concentration within ∼2 min of peptide exposure (Figure c,d). Exposure to Pal-α-MSH(6-13) and Pal-α-MSH(11-13), at 20 μM concentration, resulted in complete eradication of the MSSA cells while against MRSA, both the peptides caused 4.9 log reduction in the viable cell count. This demonstrates a direct correlation between the bactericidal activity and membrane depolarization ability of these palmitoylated peptides, suggesting that their augmented efficacy against stationary phase S. aureus cells may be the result of their enhanced membrane depolarization capability.

Figure 6

Concentration-dependent membrane depolarization of (a) MSSA and (b) MRSA cells upon peptide treatment. Survival of 106 CFU/mL dye-loaded (c) MSSA and (d) MRSA cells within ∼2 min of exposure to 20 μM concentration of the peptides (using colony count assay). Each data point represents mean ± SEM.

Upon observing the immediate membrane depolarizing potential of the palmitoylated C-terminal fragments of α-MSH, we evaluated the ability of these peptides to permeabilize the bacterial membrane by using propidium iodide (PI), a fluorogenic dye, via flow cytometry. As can be seen from the histograms shown in Figure S1a (Supporting Information) for MSSA and Figure a for MRSA, there was an apparent shift in the fluorescence of the dye from lower values to >102 arbitrary units (a.u.), indicative of PI uptake into the cells upon 1 h treatment with Pal-α-MSH(6-13) and Pal-α-MSH(11-13) at two different concentrations, namely, 10 and 20 μM, while the untreated control did not show the presence of PI positive bacterial cells. In Figure S1b (Supporting Information) and Figure b, in the presence of Pal-α-MSH(6-13) at 10 and 20 μM concentrations, 51–62% and 64–71% of the S. aureus cells showed PI uptake, respectively. Similarly, treatment with Pal-α-MSH(11-13) showed the presence of 29–33% PI positive cells at 10 μM concentration, and upon increasing the concentration to 20 μM, it permeabilized almost all the cells, i.e., 95–99% of the cells. Thus, from Figure S1 (Supporting Information) and Figure , it can be seen that although both these peptides, i.e., Pal-α-MSH(6-13) and Pal-α-MSH(11-13), appeared to be membrane-active, among the two, Pal-α-MSH(11-13) possessed superior membrane depolarizing and permeabilizing potential. This correlates well with the enhanced staphylocidal potential of Pal-α-MSH(11-13).

Figure 7

Membrane permeabilization of MRSA ATCC 33591 cells upon 1 h treatment with peptides. (a) Representative histograms showing PI uptake by cells that were (i) untreated or treated with (ii) 10 μM Pal-α-MSH(6-13), (iii) 20 μM Pal-α-MSH(6-13), (iv) 10 μM Pal-α-MSH(11-13), and (v) 20 μM Pal-α-MSH(11-13). A total of 10,000 cells were acquired for each flow cytometry analysis. (b) Percentage of bacterial cells showing PI uptake upon 1 h treatment with the peptides. Mean ± SEM from two independent experiments is presented here.

Next, we tried to determine the detrimental effects of Pal-α-MSH(6-13) and Pal-α-MSH(11-13) on the surface integrity of stationary phase S. aureus cells via scanning electron microscopy (SEM, Figure S2a in the Supporting Information and Figure a) and transmission electron microscopy (TEM, Figure S2b in the Supporting Information and Figure b). In the SEM and TEM experiments, the untreated control cells had a smooth surface and appeared as normal round cells with intact membranes. However, treatment with the palmitoylated peptides resulted in extreme alterations in the bacterial morphology and membrane architecture with severely compromised surface integrity. Several surface protrusions, blebs on the bacterial membrane, and irregular appearance of the surface could be seen in these cells. Oozing out of the intracellular material as a result of pore formation and bursting of the cells could also be observed upon treatment with the peptides. In these experiments, we used gramicidin D, a polypeptide antibiotic and well-known pore-forming agent, at 20 μg/mL as a positive control. Gramicidin D-treated cells showed the presence of cell debris, dents on the surface, and loss in shape of the cells, as reported in our earlier studies with mid-logarithmic phase S. aureus cells.[22] Thus, these microscopy observations further lend support to membrane depolarization and permeabilization being the primary mode of action of these palmitoylated peptides.

Figure 8

(a) SEM images at 50,000× magnification and (b) TEM images at 15,000× magnification of MRSA ATCC 33591 cells upon exposure to (i) no peptide, (ii) 100 μM Pal-α-MSH(6-13), (iii) 100 μM Pal-α-MSH(11-13), and (iv) 20 μg/mL gramicidin D. The arrows indicate ultrastructural changes and changes in surface morphology of the bacterial cells caused upon peptide treatment.

Antibacterial Activity of Designed Peptides in the Presence of Bacterial Growth Media

Previously, it has been shown that α-MSH and its analogues exhibit potent activity against S. aureus in physiological buffers but not in standard microbiological media.[23,25] Therefore, we wanted to examine whether, on palmitoylation, the antibacterial activity of these peptides could be restored in the presence of growth media. For this purpose, we determined the minimum inhibitory concentration (MIC) of these peptides in two different media, namely, cation-adjusted Mueller-Hinton broth (MHB) and tryptic soy broth (TSB, supplemented with 0.25% glucose and 0.5% NaCl). As seen in Table , even upon palmitoylation, α-MSH(6-13) did not inhibit bacterial growth up to the tested concentration of 45.45 μM. However, this modification significantly enhanced the activity of α-MSH(11-13) in the presence of bacterial growth media as Pal-α-MSH(11-13) exhibited potent activity against both MSSA and MRSA cells with an MIC value of 11.36 μM. Among the two conventional antibiotics used for comparison, oxacillin exhibited less activity against MRSA, while vancomycin showed potent activity against both the strains (Table ). Prior literature,[33] as well as our preliminary studies that showed that palmitic acid did not exhibit any MIC value against S. aureus cells up to the tested concentration of ∼50 μM, establish that palmitic acid alone does not show any antibacterial properties against S. aureus. Furthermore, initial studies to evaluate the serum stability of our peptide Pal-α-MSH(11-13) suggested that it is not able to exhibit similar potent activity against MRSA cells in the presence of serum (data not shown).

Table 2

Minimum Inhibitory Concentration (MIC) Values of the Palmitoylated Peptides against MSSA ATCC 29213 and MRSA ATCC 33591

	Mueller-Hinton broth		tryptic soy broth
peptide/antibiotics	MSSA	MRSA	MSSA	MRSA
Pal-α-MSH(6-13)	>45.45 μM	>45.45 μM	>45.45 μM	>45.45 μM
Pal-α-MSH(11-13)	11.36 μM	11.36 μM	11.36 μM	11.36 μM
vancomycin	0.48 μM	0.48−0.96 μM	0.48 μM	0.48−0.96 μM
oxacillin	<0.8 μM (<0.35 μg/mL)	134.1–268.3 μM (56.8–113.6 μg/mL)	<0.8 μM (<0.35 μg/mL)	134.1–268.3 μM (56.8–113.6 μg/mL)

Bactericidal Kinetics of Pal-α-MSH(11-13) in Bacterial Growth Medium against Stationary Phase S. aureus

As among the two designed peptides, only Pal-α-MSH(11-13) showed activity in the presence of bacterial growth media, we tried to determine whether its bactericidal effect was concentration- and time-dependent in TSB medium. In this experiment, higher bacterial density (107 CFU/mL) of stationary phase MSSA and MRSA cells were incubated with different concentrations of the peptide, namely, 2.5 ×, 3.5 ×, and 4.5 × MIC (i.e., 30, 40, and 50 μM, respectively), for 24 h and the results are presented in Figure .

Figure 9

Time-dependent survival of 107 CFU/mL stationary phase of (a) MSSA ATCC 29213 and (b) MRSA ATCC 33591 upon treatment with various concentrations of Pal-α-MSH(11-13) and 10 × MIC of vancomycin in the presence of TSB for 24 h. Each data point represents mean ± SEM from experiments done on 3 days.

Even at this high bacterial density, Pal-α-MSH(11-13) was able to cause ∼1.8 and 2.4 log reductions in the viability of MRSA cells at 2.5 × and 3.5 × MIC, respectively, within only 15 min of incubation (Figure b). It exerted a bactericidal effect against the MRSA cells within 1 h and 30 min of incubation at 2.5 × and 3.5 × MIC, respectively. Further, on increasing the concentration to 4.5 × MIC, Pal-α-MSH(11-13) caused a 3.4 log reduction in the cell viability within 15 min of incubation itself. Similar results were observed in the case of the stationary phase cells of MSSA (Figure a). After 24 h of incubation, 2.5 × MIC of the peptide caused a 5.2 log reduction in viability of MSSA cells while the MRSA cells exhibited a slight increase in viability, which may have occurred due to regrowth of bacterial cells in the presence of nutrient medium. At the same time, Pal-α-MSH(11-13) at 3.5 × MIC completely eradicated the MSSA cells while it caused a 5.7 log reduction in the viability of MRSA cells. At 4.5 × MIC, the peptide was able to almost completely eradicate the bacterial cells (∼7 log reduction) after 24 h of incubation in the case of both MSSA and MRSA cells. The standard comparator antibiotic, vancomycin, did not affect the cell viability even at a concentration of 10 × MIC up to 3 h of incubation, and at 24 h, it was able to cause 3.4–4.4 log reduction in the cell count.

Activity of Pal-α-MSH(11-13) against S. aureus Biofilms

After establishing the rapid and potent anti-staphylococcal activity of Pal-α-MSH(11-13) against stationary phase S. aureus cells in the presence of bacterial growth media, we further determined the efficacy of Pal-α-MSH(11-13) in inhibiting the formation of biofilm. We quantified the reduction in the biomass and metabolic activity in the biofilm via the use of crystal violet and resazurin, respectively.

As seen in Figure S3a (Supporting Information) and Figure a, at only 20 μM concentration, i.e., ∼1.7 × MIC, Pal-α-MSH(11-13) was able to completely inhibit the formation of any biomass in the case of MSSA, and for MRSA, it reduced the biomass by 98.37 ± 0.76% as compared to the untreated control biofilm, and this was comparable to the effect of vancomycin. Correspondingly, the metabolic activity in the biofilm was also reduced in the presence of 20 μM Pal-α-MSH(11-13). While the treated MRSA cells did not show any metabolic activity (Figure b), the same was reduced by 92.92 ± 0.10% for MSSA cells (Figure S3b in the Supporting Information), as seen through the resazurin assay. We also determined the survival of bacterial cells in the biofilms after peptide treatment by transferring the contents of the wells onto fresh media. At 20 μM concentration, Pal-α-MSH(11-13) also caused 4.1 and 4.2 log reductions in the survival of the biofilm-embedded MSSA and MRSA cells, respectively (Figure S3c in the Supporting Information and Figure c). Under identical conditions, comparator antibiotic vancomycin was also able to inhibit the formation of S. aureus biofilm by reducing the biomass and metabolic activity by ≥98.32 ± 0.95 and 100%, respectively, and causing a ≥4.8 log reduction in the viable cell count.

Figure 10

Inhibition of MRSA ATCC 33591 biofilm formation upon incubation with Pal-α-MSH(11-13) and vancomycin. (a) % Biomass of the biofilm quantified using crystal violet staining. (b) % Reduction in the viability of the biofilm-embedded bacterial cells as determined by the resazurin assay measuring metabolic activity. (c) Survival of the biofilm-embedded bacterial cells in log10 CFU/mL through the colony count assay. Each data point represents mean ± SEM from experiments done on 2 days. (**P ≤ 0.01).

Given the ability of our potent peptide Pal-α-MSH(11-13) to inhibit S. aureus biofilm formation, we envisaged that it could also eradicate preformed bacterial biofilms. For this, we evaluated the efficacy of Pal-α-MSH(11-13) against biofilms at different stages of maturation, i.e., a young 6 h biofilm and mature 24 h biofilm. In this context, we allowed S. aureus biofilms to grow for 6 or 24 h in 96-well plates and then exposed them to appropriate concentrations of our peptides for the next 24 h. Keeping the reduced susceptibility of bacterial biofilms in mind, the peptide concentrations used in this study, i.e., 5 ×, 10 ×, and 15 × MIC value, were higher than the concentrations tested in the biofilm formation inhibition experiment.

As seen in Figure a, Pal-α-MSH(11-13) did not exhibit any influence on the young biofilm of MSSA as there was no remarkable decrease in the biomass or viable cells. However, it was able to partially eradicate the young MRSA biofilm at even the lowest tested concentration, i.e., 5 × MIC, reducing the viable cell count by 2 log as compared to the ∼108 CFU/mL bacterial cells present in the untreated biofilm. It appears that Pal-α-MSH(11-13) exerts better efficacy against MRSA cells as compared to MSSA cells. A couple of possible explanations (e.g., different membrane compositions of MRSA cells, their fitness cost due to extra staphylococcal cassette chromosome mecA gene, etc.) may be at play here behind this observation, and for a definite answer, further exploration is required. Contrary to their effect upon young biofilms, even at the highest tested concentration, i.e., 15 × MIC, Pal-α-MSH(11-13) was unable to eradicate the mature S. aureus biofilm, and there was no appreciable decrease in either the biomass or viability of the MSSA and MRSA biofilms (Figure ).

Figure 11

Eradication of young (6 h) biofilm upon incubation with various concentrations of Pal-α-MSH(11-13). Biofilms of (a) MSSA ATCC 29213 and (b) MRSA ATCC 33591 cells were allowed to develop for 6 h and were then exposed to 5 × MIC, 10 × MIC, and 15 × MIC of Pal-α-MSH(11-13) for 24 h in TSB (0.5% NaCl, 0.25% glucose) medium. (i) % Biomass of the biofilm after treatment with the peptides quantified using crystal violet staining and (ii) survival of the biofilm-embedded bacterial cells in log10 CFU/mL through the colony count assay. Each data point represents mean ± SEM from experiments done on 2 days.

Figure 12

Eradication of mature (24 h) biofilm upon incubation with various concentrations of Pal-α-MSH(11-13). Biofilms of (a) MSSA ATCC 29213 and (b) MRSA ATCC 33591 cells were allowed to develop for 24 h and were then exposed to 5 × MIC, 10 × MIC, and 15 × MIC of Pal-α-MSH(11-13) for another 24 h in TSB (0.5% NaCl, 0.25% glucose) medium. (i) % Biomass of the biofilm after treatment with the peptides quantified using crystal violet staining and (ii) survival of the biofilm-embedded bacterial cells in log10 CFU/mL through the colony count assay.

Resistance Development Study in MRSA Cells against Pal-α-MSH(11-13)

It is believed that there is less likelihood of resistance development against membrane-active AMPs as these membranes define the phenotype, and thus, it would most probably be costly for the bacteria to generate mutations in the membrane.[11] Therefore, we evaluated the ability of MRSA to develop resistance against Pal-α-MSH(11-13) by exposing the bacteria to increasing concentrations (sub-MIC) of the peptide for several generations.[34] As seen in Figure , even after 18 serial passages, i.e., around more than 800 generations at sub-MIC concentrations, Pal-α-MSH(11-13) did not show any change in its MIC. However, under identical conditions, ciprofloxacin and vancomycin showed 66- and 8-fold increases in their MIC values, respectively. Previous studies have demonstrated a similar propensity of S. aureus strains to develop resistance against ciprofloxacin[35,36] and vancomycin.[37]

Figure 13

Development of resistance in MRSA ATCC 33591 cells upon exposure to sub-MIC concentrations of Pal-α-MSH(11-13), ciprofloxacin, and vancomycin for 18 serial passages. Each data point represents the ratio of the MIC after each passage to the MIC before the first passage.

Discussion

Bacterial biofilms are known to exhibit increased multifactorial resistance toward antibiotics and are involved in many chronic diseases.[38] Most bactericidal agents whose mode of action comprises interference with cell division or other processes necessary for cell division tend to exhibit little activity against nondividing cells and therefore biofilms.[39] Contrary to this, the usage of AMPs against bacterial biofilms holds a lot of promise as the common mechanism of action of AMPs, i.e., ability to permeabilize and/or form pores within the cytoplasmic membranes, suggests that they can exhibit high efficacy against slow-growing or stationary phase bacteria and thus against biofilm.[40] In this regard, our group is continuously trying to develop AMPs based on α-MSH.[21−28] However, the major limitation toward the therapeutic applications of these peptides is that their antibacterial activity is mitigated in the presence of bacterial growth media.[23,25,28]

Toward our ongoing efforts to overcome such limitations as well as to further improve the activity against S. aureus specifically its stationary phase, in this study, we report the design of two N-terminal lipidated analogues, by joining α-MSH(6-13) and α-MSH(11-13) with the linear fatty acid chain of palmitic acid, i.e., Pal-α-MSH(6-13) and Pal-α-MSH(11-13), respectively. Among these, Pal-α-MSH(6-13) did not show any improvement in its activity against stationary phase S. aureus cells while the staphylocidal potential of Pal-α-MSH(11-13) was significantly enhanced against the stationary phase of both MSSA and MRSA (Figure ) as compared to their parent peptides (Figure ). This difference in the ability of palmitoylation to influence the activities of these peptides may result from the structure and organization of the peptidic chains in the solution being different for the studied lipopeptides. The secondary structure of the peptides, as revealed from our CD spectroscopy study, showed that Pal-α-MSH(6-13) had a defined structure in the presence of buffer itself while α-MSH(6-13) exhibited a random coil conformation in the same environment (Figure ). Such a CD spectrum has been previously shown to indicate the existence of a β-type conformation.[41] The conformational preference of the lipidated analogue in buffer was in agreement with previous studies, which suggested that palmitoylation of peptides induced a structural transition when compared to their nonlipidated counterparts.[42] Additionally, this distinct secondary structure of Pal-α-MSH(6-13) in buffer may occur due to the interaction between the peptide and the micelles formed by the palmitic groups or oligomerization of the peptide such that the hydrophobic surfaces are packed against one another and the hydrophilic surfaces are exposed to the solution.[43] Another possibility for the observed secondary structure of Pal-α-MSH(6-13) is that the peptide may be interacting with the lipid moiety by bending on itself.[43] The blue shift observed in the Trp emission studies for Pal-α-MSH(6-13) further strengthens the possibility of the hydrophobic milieu around the Trp moiety. Encouragingly, both Pal-α-MSH(6-13) and Pal-α-MSH(11-13) showed little to no cytotoxic effect on the fibroblast cell line (Figure b), highlighting the selectivity of the peptides toward the bacterial membrane.

Similar to AMPs, lipopeptides are also known to permeate and destroy the bacterial cell membrane. Upon binding to the cell surface through electrostatic interactions, they accumulate on the surface of the cell until they reach the lipid-phase partitioning threshold and then through the microorganism’s self-promoted uptake mechanism, the lipopeptides traverse through polysaccharide barriers to reach the cytoplasmic membrane of the bacterial cells.[17,44] Similar to other AMPs and as previously reported for α-MSH and its C-terminal fragments,[21,22] the primary target of our palmitoylated peptides was the bacterial cell membrane. This was reinforced by the correlation between the staphylocidal activity of Pal-α-MSH(6-13) and Pal-α-MSH(11-13) and their ability to disrupt the bacterial membrane potential and permeabilize the membrane (Figure S1 in the Supporting Information and Figures and 7). The membrane disrupting capabilities of these two peptides were further corroborated by electron microscopy data, which also showed the highly compromised integrity of the bacterial membrane leading to the leakage of the intracellular content in the presence of Pal-α-MSH(6-13) and Pal-α-MSH(11-13) (Figure S2 in the Supporting Information and Figure ). Similar findings have been reported by other studies, which show that upon increasing peptide hydrophobicity, there is a correlated enhancement in the membrane depolarizing and antibacterial capabilities of the peptide.[45−47]

As already discussed, since medium composition is a major factor influencing the activity of α-MSH-based peptides, we studied the effect of the presence of two standard media, cation-adjusted MHB and TSB, on the antibacterial activities of our palmitoylated peptides. Like α-MSH(6-13), Pal-α-MSH(6-13) did not show activity in the presence of media as its MIC could not be determined irrespective of the medium used. However, palmitoylation markedly enhanced the activity of α-MSH(11-13), and we observed an MIC value of 11.36 μM against MSSA and MRSA in both media (Table ). Next, using the killing kinetics assay, we demonstrated the rapid and potent bactericidal effect of the most active peptide Pal-α-MSH(11–13) on the stationary phase cells of MSSA and MRSA in the presence of TSB medium (Figure ). This effect was markedly superior to the action of vancomycin, the glycopeptide currently in use against MRSA infections. It has been previously reported that the mechanism of action of vancomycin is cell wall synthesis inhibition; therefore, it typically requires 24 h and actively dividing cells to exert its bactericidal activity.[48,49] In another report, it was shown that as oritavancin, unlike vancomycin, can interact with the bacterial cell membrane, resulting in a loss of membrane integrity and collapse of transmembrane potential, it exhibited rapid bactericidal activity.[50] Similarly, the rapid bactericidal effect of Pal-α-MSH(11-13) against bacterial cells may also be attributed to it being a membrane-active peptide.

Keeping in mind the potent effect of Pal-α-MSH(11-13) against stationary phase S. aureus cells in the presence of bacterial growth media, we sought to characterize its activity against S. aureus biofilms. Pal-α-MSH(11-13) demonstrated efficient inhibition of in vitro biofilm formation for both MSSA and MRSA cells at a concentration as low as 20 μM, i.e., ∼1.7 × MIC (Figure S3 in the Supporting Information and Figure ). At the same time, similar activity was demonstrated by the comparator antibiotic vancomycin, which is known to be able to inhibit the formation of S. aureus biofilm.[51] Here, it may be noted that the biofilm inhibition efficacy of Pal-α-MSH(11-13) remains to be further explored at sub-MIC concentrations as this is an important consideration and may bring more insights into the antibiofilm activity of the peptide. Next, despite the potent inhibitory activity of Pal-α-MSH(11-13) against S. aureus biofilm as well as their activity against preformed young biofilms, it was unable to exert any statistically significant effect on mature biofilms of S. aureus (Figures and 12). Additionally, the serum stability assay revealed the reduced anti-staphylococcal potency of Pal-α-MSH(11-13) in the presence of serum-supplemented medium, which needs to be addressed in the future by further modification of the peptide.

The ability of MRSA cells to develop resistance toward membrane-active Pal-α-MSH(11-13) was determined via a multistep resistance selection study (Figure ). Encouragingly, MRSA cells did not exhibit any decrease in susceptibility toward Pal-α-MSH(11-13) over 18 serial passages, unlike the conventional antibiotics ciprofloxacin and vancomycin, which have been previously shown to develop resistance under in vitro conditions.[52,53] However, further serial passages are required to be fully confident about the degree of bacterial resistance development against this peptide. Thus, a positive implication of the current study is the low probability of resistance development against Pal-α-MSH(11-13). This may occur due to its ability to rapidly depolarize and permeabilize the bacterial membrane, which has been validated by the experimental results obtained from the PI uptake, membrane depolarization, and bactericidal kinetics assays. The quick action of the peptide against the bacteria reduces the drug exposure time required for it to develop a mechanism to counter the effect of the peptide.[54] The observed lower propensity for resistance development also hints at the existence of multiple simultaneous mechanisms of action for the peptide, which needs further exploration.

Conclusions

In conclusion, this study demonstrated that lipidation of the C-terminal fragment of α-MSH, i.e., α-MSH(11-13), not only enhanced its potency against the stationary phase of MSSA and MRSA cells but also enabled it to retain its rapid and potent staphylocidal action in the presence of bacterial growth media. The membrane disruptive mode of action of Pal-α-MSH(11-13) may also have contributed toward preventing the emergence of resistance against this analogue in MRSA cells. Most importantly, here, we also report the efficacy of Pal-α-MSH(11-13) in inhibiting the formation of biofilms of both MSSA and MRSA. The potency of Pal-α-MSH(11-13) against planktonic and sessile S. aureus suggests that its combinatorial effect with commonly used antibiotics may also be investigated as a means to mitigate antimicrobial resistance.

Materials and Methods

Chemicals

Propidium iodide (PI), crystal violet (CV), resazurin, glucose, trifluoroethyl alcohol (TFE), Triton X-100, 3,3′-dipropylthiadicarbocyanine iodide (DiSC3(5)), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DMPG), bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium (DMEM), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (USA). Fetal bovine serum (FBS) was purchased from Gibco, India. Bacterial growth media cation-adjusted Mueller-Hinton broth (MHB), tryptic soy broth (TSB, supplemented with 0.25% glucose), and tryptic soy agar (TSA) were purchased from Difco (USA). LIVE/DEAD BacLight viability assay kit was purchased from Invitrogen (Eugene, OR).

Bacterial Strains

Two bacterial strains were used in this study, viz., methicillin-sensitive S. aureus ATCC 29213 (MSSA) and methicillin-resistant S. aureus ATCC 33591 (MRSA). Both the strains were stored as 15% (v/v) glycerol stock at −80 °C until subcultured for use.

Peptides

All the α-MSH-based peptides, i.e., α-MSH(6-13), α-MSH(11-13), Pal-α-MSH(6-13), and Pal-α-MSH(11-13), were custom-synthesized from BioChain Incorporated, India. Purification of peptides (purity >98%) was performed on a semipreparative RP-HPLC column (Figure S4 in the Supporting Information), and they were characterized by LC-ESI-MS (WATERS ZQ 2000 and Agilent 6125B system; Figure S5 in the Supporting Information) by the company. Gramicidin D, vancomycin, ciprofloxacin, oxacillin, and melittin were purchased from Sigma-Aldrich (USA).

Antimicrobial Activity of the Peptides

The antistaphylococcal efficacy of the peptides in the presence of physiological buffer was determined, as described elsewhere.[28] Stationary phase bacterial cells were washed once and resuspended in 10 mM PBS (150 mM NaCl, pH 7.4). Cells were subsequently adjusted to ∼108 CFU/mL (OD600 = 0.5) and further 10-fold serially diluted to 105 CFU/mL in PBS. The cells were then exposed to the desired peptide concentrations for 2 h at 37 °C with shaking at 180 rpm. At appropriate time points, aliquots were removed, diluted, and plated on agar plates, which were then incubated overnight at 37 °C. The viable cells were determined through the counting of the colony forming units (CFU), and the data were plotted as log10 CFU/mL versus time.

Small Unilamellar Vesicle (SUV) Preparation

For the biophysical studies, namely, CD and Trp emission, artificial bacterial membrane mimic (DMPC/DMPG, 7:3, w/w) and mammalian membrane mimic (DMPC) SUVs were prepared through probe sonication, as mentioned elsewhere.[24] Initially, the two lipids DMPC and DMPG were dissolved in the chloroform/methanol mixture in a fixed ratio in a round-bottom flask. A stream of nitrogen gas was used to evaporate the solvent, and the thin lipid film obtained was dried overnight in a desiccator. The lipid film was then rehydrated with 5 mM PB and vortexed, and the suspension was swirled for 30 min in a water bath above the phase transition temperature of the lipids. The lipid dispersions were probe-sonicated for 15 min on ice (until the turbidity had cleared) using a burst time of 30 s and halt time of 10 s. Finally, the titanium debris was removed through centrifugation. The mean radius distribution of the SUVs was measured through dynamic light scattering using Xtal SpectroSize 300 (Hamburg, Germany) and was found to be in the range of 70–90 nm.

Circular Dichroism Spectroscopy of the Peptides

Circular dichroism (CD) spectra of each peptide were collected on an Applied PhotoPhysics Chirascan (Surrey, United Kingdom) instrument at 37 °C with a 1 mm path length cell.[28] In the far UV region (190–260 nm), the CD spectra of the peptides (35 μM) were acquired in 5 mM PB, helix-inducing solvent TFE (50% v/v), and DMPC/DMPG (7:3, w/w) SUVs, at 0.2 nm step size and 1 mm bandwidth. For each sample, two scans were collected at a lipid-to-peptide ratio of 41.5:1 and averaged. The spectra were analyzed after appropriate blank subtraction and expressed as ellipticity [θ] versus wavelength.

Tryptophan Fluorescence Emission Studies

Trp fluorescence emission spectra of the peptides, at 14.5 μM, in free solution, i.e., 5 mM PB, and in solutions containing DMPC/DMPG (7:3 w/w) or DMPC SUVs were obtained using a Shimadzu RF-5301 PC spectrofluorometer at a constant temperature of 25 °C.[24] The spectra were recorded in the range of 310–450 nm using a slit width of 3 nm with the excitation set at 295 nm and lipid-to-peptide molar ratio of 50:1.

Hemolytic Activity of the Peptides

The hemotoxicity of the designed peptides was determined, as mentioned elsewhere.[28] Fresh murine RBCs were washed twice with 35 mM PBS (150 mM NaCl, pH 7.4) to remove the buffy plasma coat and resuspended in the same buffer to 4% (v/v) concentration. The peptides were serially 2-fold diluted (100 μL in each well) in PBS in a 96-well plate, and the plate was incubated at 37 °C after the addition of 100 μL of the RBC suspension to every well. After 1 h incubation, the plate was centrifuged at 1500 rpm for 10 min, and 20 μL of the supernatant from each well was transferred to a different 96-well plate already containing 80 μL of PBS in every well. Using an ELISA plate reader from Molecular Devices (Sunnyvale, CA, USA), the hemoglobin release was quantified at 414 nm. 0.1% Triton X-100-treated wells served as the positive control (100% lysis), and untreated RBCs were set as the negative control. The percentage of hemolysis was determined using the formula [(OD414 of sample – OD414 of PBS)/(OD414 of 0.1% Triton X-100 – OD414 of PBS)] × 100. The assay was done in duplicate on two different days.

The present study was carried out under the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals and the Institutional Animal Ethics Committee (IAEC-02/2014) of Jawaharlal Nehru University (JNU), New Delhi, India.

Cytotoxicity Assay

The toxicity of the peptides toward the mammalian 3T3 fibroblast cell line was evaluated through the MTT assay, which is a colorimetric assay based upon the reduction of yellow MTT to insoluble, dark purple formazan crystals by mitochondrial succinate dehydrogenase of living cells. Following a protocol described elsewhere,[25] the murine cells were seeded into 24-well plates at a density of 0.2 × 105 cells/well in DMEM (containing 10% FBS) for 24 h. Once the cells reached 75% confluence, they were treated with two different concentrations of the peptides, i.e., 20 and 50 μM, dissolved in DMEM without supplementation with 10% FBS for 2 h at 37 °C in a 5% CO2 incubator. The presence of serum may lead to false negatives as the peptide may bind to the plasma protein and be less available to interact with the cells.[32] Thereafter, the wells were washed with 1 mL of PBS, 1 mL of 0.1 mg/mL MTT (in DMEM) was added to each well, and the plate was further incubated for 2 h (37 °C, 5% CO2) in the dark. DMSO (200 μL) was then added to each well to lyse the cells and solubilize the released purple formazan crystals. After transferring 150 μL of this solution to a fresh 96-well plate, the color intensity reflecting cell viability was read at 570 nm using an ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). Duplicate wells without treatment and wells treated with 2% Triton X-100 were set as growth and positive controls, respectively. The cytotoxicity of the peptides was presented as the percentage survival of cells relative to growth control calculated as (OD570 of sample/OD570 of growth control) × 100. The assay was done twice independently in duplicates.

Membrane Depolarization Assay

The cytoplasmic membrane depolarizing activity of the peptides was evaluated using the potentiometric dye DiSC3(5).[55,56] Stationary phase bacterial cell suspensions were washed, resuspended in 5 mM HEPES (20 mM glucose, pH 7.2) buffer to ∼106 CFU/mL, and preincubated with 2 μM DiSC3(5) for 30 min. Upon uptake into live cells, the fluorescence of DiSC3(5) gets quenched, and in the presence of a membrane depolarizing agent, the fluorescence detected would be higher, reflecting the release of the dye into the surrounding environment. The dissipation of membrane potential of the dye-loaded cells upon addition of increasing peptide concentrations (5 to 40 μM) was observed using a Shimadzu RF-5301 PC spectrofluorometer (Ex: 622 nm and Em: 669 nm, 3 nm slit width). The corresponding killing was also determined through a colony count assay after exposing the dye-loaded cells to 20 μM concentration of each peptide and plating aliquots onto agar plates within ∼2 min.

Bacterial Membrane Permeability Study

PI, a fluorescent intercalating agent, was used for the analysis of bacterial membrane permeabilizing ability of the peptides through flow cytometry, as described elsewhere.[57] PI is generally excluded from viable cells as the cell membrane is impervious to this dye. However, once the membrane is disrupted, PI enters the cell and fluoresces upon binding to double-stranded DNA. Briefly, S. aureus cells were grown in TSB (0.25% glucose, 0.5% NaCl) overnight until they reached the stationary phase, harvested, washed, and then resuspended in 10 mM PBS (150 mM NaCl, pH 7.4) to a final density of 105 CFU/mL. The cells were then incubated with two concentrations of the peptides, i.e., 10 and 20 μM, for 1 h at 37 °C with shaking at 180 rpm. PI at 1.3 μg/mL was then added to the cells and kept at 37 °C in the dark for the last 20 min of peptide treatment. After incubation, the samples were excited at 544 nm, and the PI fluorescence was recorded at an emission wavelength of 620 nm using Becton Dickinson (BD) FACSverse (San Jose, CA). In this experiment, cells with >102 a.u. of fluorescence were considered to have taken up PI.

Scanning Electron Microscopy (SEM)

Bacteria of the stationary phase were diluted to an OD600 of 1.0, i.e., ∼109 CFU/mL, in 10 mM PBS (150 mM NaCl, pH 7.4) and treated for 2 h with 100 μM concentration of the peptides, due to the high bacterial inoculum required for microscopy, at 37 °C with shaking at 180 rpm. Bacterial cells treated with 20 μg/mL of a well-known membrane disrupting linear pentadecapeptide, gramicidin D, served as a positive control in this experiment. Untreated bacterial cells were prepared as the growth control. After 2 h, the cells were centrifuged, and the pellets were washed thrice with PB (pH 7.4) and kept at 4 °C for overnight fixation with 2.5% glutaraldehyde (in PB). Next, the cells were washed three times with the same buffer, followed by dehydration with a graded series of ethanol (30–100%). The dehydrated samples were dried in a vacuum desiccator, and after coating them with gold particles (20 nm), the samples were observed under a scanning electron microscope (EVO 40, Carl Zeiss, Germany).[22]

Transmission Electron Microscopy (TEM)

TEM of the bacterial samples was performed according to a protocol described elsewhere with slight modifications.[22] The samples were processed as described for SEM sample preparation. After overnight fixation of the control or treated specimens with 2.5% glutaraldehyde at 4 °C, the cell suspensions were also fixed with 1% osmium tetroxide and sequentially dehydrated by graded acetone series (50–100%). The samples were embedded in epoxy resin, and ultrathin sections were prepared using a microtome. After placing these sections onto a copper grid, they were stained sequentially with uranyl acetate and lead citrate. The samples were then washed and dried under vacuum in a desiccator. TEM analysis was performed with a JEOL JEM 2100 (Japan) microscope at 120 keV electron energy.

Determination of Minimum Inhibitory Concentration (MIC) of the Peptides

For the experiment, a previously defined serial broth microdilution method was used with slight modifications.[58,59] Briefly, mid-logarithmic phase bacterial cells from the secondary culture were washed, resuspended in cation-adjusted MHB or TSB (0.25% glucose, 0.5% NaCl), and spectrophotometrically adjusted to 108 CFU/mL (OD600 of 0.5). In a polypropylene 96-well plate (Corning Incorporated, USA) containing 10 μL of serially 2-fold diluted peptides or antibiotics in 0.2% BSA (in 0.01% acetic acid), 100 μL of the bacterial suspension was added at a final density of ∼2–5 × 105 CFU/mL in each well. To one column of the microtiter plate, only media were added (negative control), and another column contained bacterial cells without any treatment (growth control). The plates were incubated at 37 °C with shaking at 180 rpm for 16–18 h, and the absorbance at 600 nm was recorded using an ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). The lowest concentration of the peptide/antibiotic completely inhibiting the bacterial growth, as observed spectrophotometrically, was taken as the MIC of that agent. The experiment was done on at least two different days in duplicate. The stability of the peptide in the presence of serum was also determined through the MIC study by diluting FBS in TSB medium to a final concentration of 25% (v/v) and then preparing the bacterial inoculum in the FBS-containing medium.

Bacterial Killing Kinetics Assay in Microbiological Medium

To determine the potential of the designed peptide against the stationary phase of S. aureus in the presence of standard microbiological media, a standard drop plate method was used.[58,60,61] Briefly, S. aureus was grown to stationary phase in TSB (0.25% glucose, 0.5% NaCl) at 37 °C on an orbital shaker (180 rpm) and adjusted to 107 CFU/mL (OD600 of 0.05) in the same medium. Subsequently, the samples were incubated with three different concentrations of Pal-α-MSH(11-13), i.e., 30, 40, and 50 μM, at 37 °C. Standard comparator antibiotic vancomycin was also included in this experiment. At different time points of incubation, viz., 15 min, 30 min, 1 h, 2 h, 3 h, and 24 h, aliquots of the samples were removed, serially diluted, and plated on agar plates. After incubating the plates at 37 °C for 16–18 h, the viable cell count was determined by counting the colonies. The experiment was performed thrice independently on different days.

In Vitro Efficacy of Peptides against the Bacterial Biofilm

To evaluate the antibiofilm potential of our peptide, we determined its ability to inhibit the formation of biofilms as well as eradicate mature staphylococcal biofilms. For the biofilm formation inhibition experiment, biofilms were grown for 24 h in the presence of test agents following a previously defined protocol with slight modifications.[35,56] Briefly, overnight grown S. aureus (∼108 CFU/mL) was diluted in fresh TSB (0.25% glucose, 0.5% NaCl) medium to the desired inoculum size (105 CFU/mL). One-hundred microliters of this bacterial suspension was dispensed in a polystyrene 96-well plate, and the biofilm was allowed to grow for 24 h at 37 °C in the presence of an appropriate concentration of the peptide, i.e., 20 μM. The growth control did not have any peptides added to the wells, and to the negative control wells, only 100 μL of TSB media was added. After 24 h, the biofilms formed in the wells were evaluated using crystal violet (CV) staining for determination of the biofilm biomass, resazurin assay for evaluation of the metabolic activity within the biofilm, and colony count method to quantify the biofilm-embedded bacterial cells.

The ability of the peptide to eradicate biofilms was also evaluated against established biofilms of S. aureus, including both young (6 h) and mature (24 h) biofilms, using an earlier described protocol[56] with slight modifications. Initially, an overnight culture of S. aureus was diluted in fresh TSB medium to an inoculum size of ∼105 CFU/mL. One-hundred microliters of this bacterial suspension was dispensed in the wells of a polystyrene microtiter plate and incubated at 37 °C without shaking for 6 or 24 h. The negative control wells contained uninoculated medium. After the respective incubation periods, the biofilms were washed once with 200 μL of 10 mM PBS (pH 7.4), and 100 μL of fresh TSB medium containing desired concentrations of the peptides was added to the wells. Fresh TSB medium without any agents was added to the growth control and negative control wells. The 96-well plates were incubated for another 24 h at 37 °C without shaking after which the biofilms present in the wells were processed through CV staining and colony count assay.

Determination of Biomass of the Biofilm through Crystal Violet Staining

Crystal violet is used for evaluating the biomass of the biofilm as it is a known cationic dye that nonspecifically stains the negatively charged biofilm constituents, i.e., the live and dead cells as well as the extracellular polymeric substances.[62] In this assay, after the respective incubation periods, the spent medium was removed from the microtiter plates used in the biofilm experiments, and the wells were washed twice with PBS gently. After that, the biofilms formed in the wells were heat-fixed at 60 °C for 1 h and stained for 20 min with 125 μL of 0.1% aqueous CV solution. The wells were washed twice with PBS gently to remove the excess CV and the dye that still adhered to the biomass of the biofilm was dissolved in 125 μL of 33% glacial acetic acid. The plate was then read at 570 nm to quantify the biomass of the biofilm.

Determination of Metabolic Activity within the Biofilm through Resazurin Assay

To get a better sense of the viability of the bacterial cells within the biofilm, the metabolic activity of the biofilm-embedded cells was determined using resazurin. This blue-colored phenoxazin dye in the presence of viable cells gets readily reduced to resorufin, a pink fluorescent compound.[63] For the assay, the biofilm-containing microtiter plates were washed twice with 200 μL of PBS and 10 μg/mL resazurin in TSB was added to each well. The plates were sonicated for 5 min at 40 kHz to remove adhered cells and kept at room temperature in the dark for 30 min. After 30 min, plates were read using an excitation wavelength of 550 nm and emission was collected at 590 nm.[64]

Determination of Viable Bacterial Cells within the Biofilm through Colony Count Assay

The microtiter plates used in the biofilm experiments were washed with 200 μL of PBS, and to each well, 100 μL of PBS was added. The wells were scraped meticulously and contents were transferred to fresh microcentrifuge tubes. The cell suspensions were sonicated for 5 min at 40 kHz and plated on agar plates, and the viable colony forming units were counted after incubating the plates at 37 °C for 16–18 h.[56]

In Vitro Resistance Development Study

The development of resistance in MRSA was monitored through a serial passage study, as described elsewhere with slight modifications.[35,56] Briefly, in this multipassage experiment done in 96-well plates as a series of individual MIC experiments, initially, a single bacterial colony was inoculated in cation-adjusted MHB medium and grown overnight at 37 °C. On the first day of the experiment, the 96-well plate containing serial dilutions of Pal-α-MSH(11-13), ciprofloxacin, and vancomycin (as previously described in the MIC protocol) was seeded with ∼2–5 × 105 CFU/mL mid-logarithmic phase bacterial cells and incubated at 37 °C overnight. The next day, MIC was determined using the MIC protocol as described earlier, and 100 μL of the cell suspension from the wells with concentrations 2–4 fold less than the MIC value for the respective drug was taken as inoculum for the next day’s MIC assay. This process was repeated for a total of 18 passages, and aliquots of the bacterial suspensions from each day were stored at −80 °C as 15% (v/v) glycerol stocks.

Statistical Analysis

GraphPad Prism 5 software was used for the calculation of statistical significance and one-way analysis of variance with post hoc Bonferroni’s test was applied. A P value of <0.05 was considered to be statistically significant.