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Membrane-Active Amphipathic Peptide WRL3 with in vitro AntibiofilmCapability and in vivo Efficacy in Treating MRSA Burn Wound Infections Zhi Ma, Jinzhi Han, Bingxue Chang, Ling Gao, Zhaoxin Lu, Fengxia Lu, Haizhen Zhao, Chong Zhang, and Xiaomei Bie ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00100 • Publication Date (Web): 08 Sep 2017 Downloaded from http://pubs.acs.org on September 9, 2017

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ACS Infectious Diseases

Membrane-Active Amphipathic Peptide WRL3 with in vitro Antibiofilm Capability and in vivo Efficacy in Treating MRSA Burn Wound Infections Zhi Ma†, Jinzhi Han†, Bingxue Chang, Ling Gao, Zhaoxin Lu, Fengxia Lu, Haizhen Zhao, Chong Zhang, Xiaomei Bie* College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

†

These authors contribute equally to this work.

*Correspondence: E-mail: [email protected] (X. Bie) Postal address: 1 Weigang, Xuanwu District, Nanjing, China 210095

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Methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly prevalent in hospitals, clinics, and the community. MRSA can cause significant and even lethal infections, especially in skin burn wounds. The currently available topical agents have largely failed to eliminate MRSA infections due to resistance. Therefore, there is an urgent need for new and effective approaches for treating MRSA. Here, we show that a novel engineered amphipathic peptide, WRL3, exhibits potent antimicrobial activity against MRSA, even in the presence of various salts or serum. The cell selectivity of WRL3 was demonstrated by its ability to specifically eliminate MRSA cells over host cells in a co-culture model. Additionally, WRL3 showed a synergistic effect against MRSA when combined with ceftriaxone, and effectively inhibited sessile biofilm bacteria growth leading to a reduction in biomass. Fluorescent measurements and microscopic observations of live bacterial cells and artificial membranes revealed that WRL3 exerted its bactericidal activity possibly by destroying bacterial membrane. In vivo studies indicate that WRL3 is able to control proliferation of MRSA in wound tissue and reduce bioburden, and provides a more favorable environment for wound healing. Collectively, our data suggest that WRL3 has enormous potential as a novel antimicrobial agent for the treatment of clinical MRSA infections of skin burn wounds. Keywords: Antimicrobial peptides, Bactericidal mechanism, Cell selectivity, MRSA burn wound infection

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Staphylococcus aureus (S. aureus) is a remarkably versatile and opportunistic pathogen that has confounded the medical community by its ability to resist antibiotics. It is the most common cause of skin infections and the second most common cause of nosocomial bloodstream infections.1-2 Strains of S. aureus that exhibit resistance to all currently available β-lactam antibiotics, including penicillins and cephalosporins, are commonly referred to as methicillin-resistant S. aureus (MRSA).3 When MRSA enters the body through an abrasion or burn wound, it may cause serious invasive diseases, such as sepsis, endocarditis, toxic shock syndrome, and necrotizing pneumonia, by evading the natural protective mechanisms of the body.4 The presence of biofilms is a key factor in delaying wound healing and further complicates the treatment of infectious diseases. Biofilm formation, which occurs when microbial cells adhere to each other and become embedded in a matrix of extracellular polymeric substance (EPS) on a biotic or abiotic surface, has been implicated in a variety of chronic microbial infections, such as osteomyelitis and pyomyositis5. The eradication of biofilms presents an immense medical challenge due to the poor penetration of antibiotics through the EPS matrix and/or their inactivation by components of the EPS matrix, which often results in the rapid development of antibiotic resistance6. Currently, vancomycin is the first-line therapy for the treatment of MRSA infections. However, the emergence of vancomycin-resistant S. aureus strains highlights the need for new classes of antimicrobials with completely different modes of action7. The mounting crisis of antibiotic-resistant infections, together with the ongoing dearth in new small-molecule antibiotic development, have spurred considerable efforts towards the discovery and development of membrane-active antimicrobial peptides (AMPs) as alternative antimicrobials8. Compared with conventional antibiotics, AMPs possess

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multiple modes of action, rapid kill kinetics, broad-spectrum antimicrobial activity, and minimal host toxicity9. Generally, AMPs do not have specific microbial cell targets; instead, the bacterial membrane is considered to be the main target of AMPs. It is precisely because of this mechanism that bacteria have a lower potential for developing resistance to AMPs, as the entire membrane structure or membrane lipid composition of a microbe would need to be changed.10 In addition to decreasing the emergence of resistance, some natural AMPs are also involved in the modulation of the immune response. For instance, TP4, a 23 amino acid peptide, induces a Th1 cellular immune response and acts as an adjuvant to vaccines in tilapia.11 Clinical case studies have shown that application of AMPs to cutaneous wounds can both help reduce the risk of microbial infection and reduce the overall time required for wound healing.12 A recent study also reported that AMPs may promote resistance to bacterial infections by stabilizing the cytoskeletal network in host cells.13 Recently, we designed a series of amphiphilic α-helical peptides based on Leucocin A by substituting non-charged hydrophilic residues with arginine and leucine. Of the engineered peptides, WRL3 (WLRAFRRLVRRLARGLRR-NH2) exhibited the highest cell selectivity towards bacterial cells over erythrocytes and macrophages. Moreover, WRL3 demonstrated potent antimicrobial activity in vitro against methicillin-resistant Staphylococcus aureus, with the minimum inhibitory concentration of 2 µg/ml.14 However, the efficacy of WRL3 as a novel antimicrobial in treating MRSA wound infections in vivo has not been thoroughly studied. In this study, we further evaluate the in vitro antimicrobial activities of WRL3 against MRSA in the absence or presence of cations and serum. Synergistic effects of WRL3 and conventional antibiotics were examined by the checkerboard titration method. The cell selectivity of WRL3 was assayed in a co-culture of MRSA and host cells, with the killing efficacy

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assessed using a time-kill kinetic assay. Atomic force microscopy (AFM), transmission electron microscopy (TEM), confocal microscopy, flow cytometry, the localization of fluorescently labelled peptide in model membranes, and dye leakage assays were performed to help understand the bactericidal mechanism of WRL3 against MRSA. The effectiveness of WRL3 in eradicating the preformed biofilms of MRSA was determined in vitro. Furthermore, the effects of WRL3 (alone or mixed with ceftriaxone) on removal of bacteria in wounds, pro-inflammatory cytokine secretion, macrophage and monocyte recruitment, angiogenesis, and wound healing in MRSA-infected mice were examined using ELISA, histological, and immunohistological approaches. In summary, the results reported here suggest that WRL3 may be suitable for use as a novel effective antimicrobial for the treatment of multi-drug resistant S. aureus infections. ■ RESULTS Antimicrobial Activity. The antimicrobial activities of WRL3 and antibiotics against MRSA and various clinically isolated S. aureus were summarized in Table 1. Overall, WRL3 displayed potent antibacterial activity, with a geometric mean (GM) MIC of 4 µg/mL, comparable to polylysine (GM = 4.9 µg/mL), ceftriaxone (GM = 3.0 µg/mL), and methicillin (GM = 5.2 µg/mL). However, WRL3 had a lower antibacterial activity against S. aureus than vancomycin (GM = 0.9 µg/mL), gentamicin (GM = 0.6 µg/mL), and ciprofloxacin (GM = 0.7 µg/mL). Notably, WRL3 displayed higher antimicrobial potency against MRSA and clinically isolated multidrug-resistant strains than the antibiotics tested in this study (except for vancomycin and ciprofloxacin). To further investigate the ability of WRL3 to kill MRSA, we determined its lethal concentration (LC), which is defined as the lowest concentration of an antimicrobial agent that completely kills the bacteria. We found that WRL3 completely killed MRSA

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cells at the concentration of 4 µg/mL, and displayed approximately 8- to 128-fold higher bactericidal activity than the antibiotics tested (Table 2). To further understand the efficiency of WRL3 in killing MRSA, the time-kill kinetics of WRL3 and antibiotics

interaction

with

MRSA

at

multiple

concentrations

were

investigated

in

the

phosphate-buffered saline (PBS) solution (10 Mm, pH 7.4). WRL3 induced time-dependent growth inhibition of MRSA and eliminated almost all MRSA cells within 5 min, even at the lowest tested concentration (1 × MIC, 2 µg/mL), indicating a rapid and substantial killing efficacy (Figure 1). We also observed a similar trend for nisin; however, even at the highest concentration (4 × MIC, 512 µg/mL), a complete kill occurred in 60 min, suggesting a lower bactericidal efficiency than WRL3. MRSA appeared to be less sensitive to the other antibiotics tested, which exhibited minimal or no bactericidal effects at all the concentrations tested within the 120 min time period. As a whole, WRL3 showed the fastest and most remarkable killing efficiency against MRSA among the selected antimicrobial agents in this study. Synergy with Antibiotics. To investigate potential synergy between our peptide and conventional antibiotics, the antibacterial activities of combinations of WRL3 and antibiotics against MRSA were analyzed by the checkerboard assay. WRL3 displayed synergistic effects with ceftriaxone, with a fractional inhibitory concentration index (FICI) value of 0.375 against MRSA (Table 3). In combination with other antibiotics, WRL3 showed either additive effects or no interaction, with FICI values ranging from 0.625 to 1.5. Salt and Serum Sensitivity. To investigate whether the antimicrobial activities of WRL3 and antibiotics were compromised in the presence of cations, the MIC values of peptide and antibiotics

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were measured in various salt conditions. The addition of monovalent (Na+, NH4+, and K+) and divalent (Ca2+, Mg2+, and Zn2+) cations had only a slight repressive effect or even promoted bacteriostatic activities of WRL3, with MICs ranging from 0.5 to 4 µg/mL (Table 4). With the exception of vancomycin and ciprofloxacin (which maintained their strong activities against MRSA in the presence of various salt ions), the conventional antibiotics appeared to be more susceptible to the addition of monovalent and divalent cations compared with WRL3; they showed 4- to 16-fold increases in MIC values. Noticeably, the most significant loss in antibacterial activity was observed for the peptide and antibiotics in the presence of Fe3+. To further determine the effect of physiological conditions on the antimicrobial activity of the peptide, MRSA cells were treated with increasing peptide concentrations in the presence of serum (Table 4). Overall, the antimicrobial activities of WRL3, vancomycin, and ciprofloxacin were only slightly compromised or even increased with the addition of serum. In contrast, the MICs of the other antibiotics greatly increased with the addition of serum, with the most significant losses noted for polylysine and gentamicin in the presence of 50% serum. Together, these data suggest that the activity of WRL3 might be particularly beneficial in physiological conditions for the treatment of MRSA infections in vivo. Cell Selectivity. We found that even at the peptide concentration of 64 µg/mL, WRL3 exhibited minor or no toxicity to erythrocytes and human liver L-O2 cells (Table 2). To further investigate the ability of WRL3 to discriminate bacterial cells from mammalian cells, a co-culture model where liver L-O2 cells were infected with MRSA prior to peptide treatment was utilized. As shown in Figure 2A, WRL3 decreased the bacterial load by 85% within 30 min and eliminated total extracellular MRSA cells

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within 120 min. A similar trend was observed for vancomycin, but the bacterial cells were not completely eradicated within 150 min. By comparison, methicillin displayed almost no activity against MRSA cells in co-culture with liver cells. We also measured eukaryotic cell viability using a 3-(4, 5-dimethylthiozol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay; neither WRL3 nor antibiotics affected the viability of liver L-O2 cells (0% cytotoxicity) at 1 × MIC (Figure 2B). The high cell selectivity indicates that WRL3 could be an effective and safe therapeutic agent. Mechanism of Action of WRL3 against MRSA. We also evaluated the antimicrobial mechanism of WRL3 by determining the integrity of bacterial membranes after treatment. Propidium iodide (PI) was used to fluorescently stain the nucleic acids of cells when the cytoplasmic membrane integrity was disrupted. With no peptide treatment, 93.8% of the bacterial cells demonstrated no PI fluorescence, suggesting that the bacterial cytoplasmic membranes were intact (Figure 3A). However, the addition of WRL3 induced a significant increase in fluorescence intensity (Figure 3H-J), indicating severe damage to membrane integrity. The same experiment with methicillin or vancomycin in place of WRL3 did not increase fluorescence. These results indicate that WRL3 is capable of destroying the MRSA cell cytoplasmic membrane, leading to cell death. To further elucidate the antimicrobial mechanism of WRL3 against MRSA cells, AFM was employed to study morphological alterations.15 With no peptide treatment, MRSA cells had smooth membrane surfaces (Supplemental Figure S1A); however, after treatment with WRL3 at 1 × MIC for 30 min, membrane atrophy, creping, and breakage were observed in MRSA cells (Supplemental Figure S1F). After the cell surfaces were exposed to the peptide for 60 min, they became completely roughened and corrugated, and exhibited atrophy and fractures (Supplemental Figure S1G). In comparison, the cell

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membrane surfaces treated with methicillin and vancomycin showed either no or minor changes, and were similar to the untreated MRSA cells. TEM images of MRSA cells incubated with the peptide showed that WRL3 induced distinct rupture of the cytoplasmic membrane after 30 min of exposure; after 60 min, the complete collapse of the cytoplasmic membrane and leakage of the intracellular contents induced by WRL3 were observed (Figure 4F and G). In contrast, there was no damage to the bacterial membranes or release of intracellular contents following 60 min treatments with methicillin or vancomycin. These observations are in agreement with widely accepted models of cationic peptide disruption of the cytoplasmic membrane.8, 16 To precisely locate the site targeted by the engineered peptide during incubation with MRSA cells, the fluorescence distribution of fluorescein isothiocyanate (FITC)-labeled WRL3 (2 µg/mL) was visualized using confocal microscopy at different points during incubation. As shown in Figure 5, after 15 min of incubation, bacterial cells treated with peptide WRL3 presented green circular rings with fluorescent signals spread across the bacterial membrane surfaces, indicating that the membranes surrounding the cells are the primary targets during this period. After 30 min of incubation, the fluorescent signals clustered in the cytoplasm, suggesting the translocation of the peptide across the lipid bilayers. We investigated the interaction of FITC-labelled WRL3 with negatively-charged phosphatidylglycerol (PG)/cardiolipin (CL) (3:1, w/w) liposomes, which mimic S. aureus membranes, to gain insight into the effects of WRL3 on the MRSA membrane. At 0 min, the vesicles have not yet encountered WRL3 (Figure 6A). After incubation with 2 µL of WRL3 for 10 min, the fluorescent signals started to diffuse into

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the solution and the vesicles were partially stained by FITC-labelled peptide (Figure 6B). After 30 min of incubation, a brilliant green circular ring could be seen (Figure 6C), indicating that WRL3 was gathered at the vesicle's surface. After 45 min of incubation, the fluorescence intensity on the surface decreased slightly and a fluorescence signal was visible inside the vesicles (Figure 6D). A liposome leakage assay was used to determine if WRL3 exerts antimicrobial activity by pore formation and/or membrane perturbation. As indicated in Figure 7A-C, unlike methicillin and vancomycin, WRL3 dose-dependently induced calcein leakage from the negatively-charged PG/CL liposomes, indicating that WRL3 caused the destruction of bacterial membranes by pore formation. Interestingly, phosphatidylcholine

(PC)/cholesterol

(CHO)

(10:1,

w/w)

liposomes

(a

mammalian

membrane-mimicking environment) showed no leakage on treatment with WRL3 or antibiotics (Figure 7D). This further confirms the cell selectivity of WRL3 towards the negatively-charged bacterial membranes rather than zwitterionic mammalian cell membranes. Antibiofilm Activities. We next determined the antibiofilm activities of WRL3 and antibiotics on preformed biofilms, which are intrinsically more challenging than the prevention of biofilm formation.17 As indicated in Figure 8A, all the antimicrobials displayed a dose-dependent inhibition of MRSA growth in biofilms. WRL3 was found to cause a more significant decrease than the antibiotics in viability of bacterial cells across the five concentrations tested, with cell viability reduced to < 30% at 8 × MIC within 24 h. To investigate the relative amounts of biomass remaining after treatment with the peptide and antibiotics, the biofilms were stained with crystal violet and solubilized. The amount of biomass in the treated biofilms decreased in a dose-dependent manner, similar to the decrease in MRSA cell viability (Figure 8B). The changes in the amount of biomass after treatment with WRL3 were further

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examined by using scanning electron microscopy (SEM). MRSA cell densities in the biofilms were substantially reduced with increasing peptide concentrations (Figure 8C), and there was a remarkable disruption in biofilm cell surfaces at 1 × MIC. This observation is consistent with the reductions in cell viability and biomass. Thus, we provide direct evidence that WRL3 is able to kill microbes embedded within biofilms and efficiently disintegrate the preformed biofilms. The in vivo Efficacy of WRL3 in MRSA-infected Burn Wound Model. Methicillin treatment displayed no inhibitory growth effects on MRSA cells in burn wounds. However, MRSA CFUs in burn wounds treated with WRL3 was significantly decreased (to 60%) during the initial 3 days of treatment and they were almost completely eradicated by the 14th day. A similar trend was observed with vancomycin, but it was weaker than that for WRL3 (Figure 9A). Figure 9B showed the area of the burn wound region, which was measured daily in uninfected mice (control), and infected mice treated with methicillin, vancomycin, WRL3, ceftriaxone or WRL3 and ceftriaxone. The size of the healed area was compared to that of the initial wound to assess wound healing efficiency. The data showed that almost all of the wounds from the treatments with WRL3, vancomycin, and WRL3+CRO were nearly closed by day 17, suggesting that the in vivo efficacy of WRL3 is comparable to vancomycin. The burn wounds festered on the mice that were infected with MRSA (Figure 9C). In contrast, the uninfected mice showed normal burn wounds that did not fester (control). In the wounds treated with WRL3 in combination with ceftriaxone, the scabs started to detach from the wound on the 7th day after burning and completely detached on the 12th day. This was similar to the control group. The scabs of burn wounds treated with vancomycin and WRL3 began to detach from the wounds on the 14th and 11th days and had completely fallen off by the 20th and 16th days, respectively. These results indicate that

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WRL3 is more efficient than vancomycin in reducing MRSA infections and that it shortens the healing time of burn wounds. Moreover, the co-treatment of MRSA-infected burn wounds with WRL3 and ceftriaxone enhanced wound healing compared with treatment with WRL3 alone (Figure 9C). As a whole, bacterial counts decreased more rapidly and wound healing was faster in burn wounds treated with WRL3 than in wounds treated with vancomycin. The effect of WRL3 on the immune response induced by MRSA was also examined. MRSA infection resulted in high levels of inflammatory cytokines (IL-6, IL-10, and TNF-α) in the serum on the first day post-infection (Figure 10A). As expected, methicillin treatment only mildly suppressed pro-inflammatory cytokine generation. In contrast, treatment with vancomycin or WRL3 (in the presence or absence of ceftriaxone) significantly decreased the production of IL-6, IL-10, and TNF-a. The chemoattractant MCP-1 is involved in the recruitment of monocytes to the site of injury, and helps traffic proteins across the endothelial barrier during wound healing. MCP-1 production, which is induced by MRSA infection, was markedly decreased following treatment with WRL3 and ceftriaxone; however, it was not affected by treatment with either vancomycin or WRL3 alone. The wounded regions were subjected to Gram staining (Figure 10B); the purple staining of Gram-positive MRSA was completely absent in infected mice treated with WRL3 alone, or WRL3 mixed with ceftriaxone. These results provide additional confirmation that WRL3 can effectively inhibit MRSA infections in skin burn wounds. During injury recovery, angiogenesis occurs to supply blood to the wounded region. Angiogenesis efficiency was also compared between the various treatment groups through a light microscopy (Figure 10C) and HE examination (Figure 11). The results showed that more new blood vessels were observed following treatment with WRL3 than treatment with vancomycin in infected mice.

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Histology and Chemotaxis of Burned Tissue. As shown in Figure 11, normal skin (the blank control) can be divided into epidermal (a), dermal (b), and subcutaneous adipose (c) layers. In the normal skin samples, the layers were complete and clearly bounded. Compared with the burn control, the MRSA-infected wound sample showed a serious inflammatory reaction, and the epidermis was severely damaged between post-burn days 3 and 14. Similar findings were seen in methicillin-treated wounds. In contrast, the WRL3-treated sections harvested on post-burn day 3 showed a thickened epidermis (d) at the wound margin and in the underlying viable hair follicles (arrows in Figure 11). Epithelial cells from superficial hair follicles formed a portion of the advancing epidermal layer (arrowhead in Figure 11). On post-burn day 7, the wound showed active re-epithelialization with multiple islands of epithelial cells migrating to the wound surface from the underlying dermal appendages. Complete wound re-epithelialization was observed on post-burn day 14, with a thickened epidermis (e) and reactive hair follicles (arrows in Figure 11) beneath the burn surface. There was a relatively slower wound re-epithelialization with vancomycin treatment than with WRL3 treatment. We also found that WRL3 in combination with ceftriaxone was more effective at improving the re-epithelialization of scald injuries infected with MRSA compared with vancomycin or WRL3 treatment (Figure 11). To remove wound debris during healing, monocytes begin to replace neutrophils at 48 h; this is followed by induction of several growth factors during the proliferation phase after 72 h. Tissue sections from the wounded area were stained and probed with specific antibodies for monocytes, macrophages and vascular endothelial growth factor (VEGF). MRSA infected wound samples revealed a large accumulation of macrophages and monocytes (Supplemental Figure S2A and B); but WRL3 treatment

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reduced their recruitments. Production of VEGF is important for provisioning the required oxygen, nutrients, and immune cells for antibacterial activity and wound healing. Staining against VEGF was increased in infected sections treated with WRL3 compared with those treated with vancomycin (Supplemental Figure S2C). ■ DISCUSSION AND CONCLUSION S. aureus is a serious challenge during treatment of infections, and selection of the most appropriate antimicrobial is typically complicated. The efficacy of the currently available anti-staphylococcal agents is frequently compromised by the ability of S. aureus to develop resistance to multiple classes of antimicrobials.18-19 The emergence of methicillin-resistant S. aureus (MRSA) makes S. aureus infections more difficult to eliminate or control with existing antibiotics. An urgent but arduous task is to effectively eradicate this opportunistic bacterial pathogen or slow the emergence of resistance to available drugs.20 Our data demonstrated that WRL3 had potent bacteriostatic (Table 1) and bactericidal (Table 2) activity against various S. aureus strains, even against MRSA and multidrug-resistant clinical isolates. Interestingly, although the antibiotics displayed greater bacteriostatic activities, they had relative lower bactericidal activities against MRSA cells within 120 min (Table 2). Previous reports have shown that the antimicrobial mechanism of most cationic AMPs acts through physical disruption of bacterial membranes, which causes intracellular content release and eventually leads to bacterial cell death.16,

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Generally, antibiotics inhibit microbial proliferation by

intervening cell wall or protein synthesis; however, they do not appear to destroy the synthesized cell wall/membrane and directly kill the bacterial cells. This is one explanation for why WRL3 exerts potent bactericidal activity in PBS, but the antibiotics do not. Moreover, time-kill curve analysis demonstrates

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that, compared with the antibiotics, WRL3 exhibits a rapid killing efficiency against MRSA cells. This rapid and effective peptide clearance in a host environment contributes to shortening the duration of antimicrobial treatment and decreasing the occurrence of antibiotic-resistant genetic mutations or phenotypic variants, known as biofilms or persister cells.22 Monotherapy with specific agents against S. aureus usually results in the development of resistance.23 For instance, in vitro studies found that rifampicin monotherapy invariably led to the emergence of resistant mutants of S. aureus; however, this can be suppressed by the addition of a quinolone.24 Therefore, a combination of antibacterial drugs may be the simplest and most effective strategy used for treating MRSA infections. As reported previously, synergistic effects among cationic AMPs and conventional antibiotics can improve antibacterial activity.25 This effect is attributed to the enhanced intracellular access of the drug aided by membrane-permeabilizing peptides.26 Our data showed that WRL3 exhibited synergistic action with ceftriaxone against MRSA. Although there was no synergistic effect between WRL3 and other antibiotics, the peptide substantially decreased the MIC values of the antibiotics, indicating that, in combination, WRL3 could potentially be used to decrease the dose of antibiotics required for clinical applications. Many studies have showed that the presence of cations, such as Na+, can adversely influence the antibacterial activity of natural AMPs.27 In this study, polylysine displayed a high sensitivity to cations, with a 2- to 16-fold increase in MIC values following the addition of various salt ions. Electrostatic interactions between cationic portions of the peptide and the negative-charge on the bacterial surface facilitate the interaction between the peptide lipophilic regions and the bacterial membrane, leading to membrane disruption and cell death.28 Therefore, it is not difficult to infer that any factor which reduces

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this electrostatic interaction, such as a higher ionic strength in solution, may prevent AMPs from binding to the bacterial membrane and result in decreased antimicrobial activity. Cationic AMPs often show decreased activity in the presence of other cations,27 but this is not always the case. The antibacterial activity of WRL3 was only slightly compromised or even increased in physiological salt conditions. This underlying salt resistance mechanism of WRL3 can be attributed to its high net charge and the extremely amphipathic structure of the molecule. The net charge of WRL3 is +9; thus, this high cationicity could help to overcome the inhibitory effects of monovalent cations, which normally interrupt the electrostatic attraction between positively-charged peptides and negatively-charged membranes.29 In addition, WRL3 displays highly amphipathic characteristics, with hydrophilic residues on the polar side chain and hydrophobic residues on the nonpolar side chain; this leads to a stronger affinity for the bacterial membrane.30 Moreover, this amphipathic characteristic further reinforces the stability of α-helical structure, which can resist the counter effect of divalent or multivalent cations competing for membrane binding with peptides.31 The resistance of WRL3 to salts could be critical to the treatment of infections in diseases that might disturb normal salt homeostasis in certain human tissues. Previous reports have shown that natural AMP activity is usually suppressed in serum, which has greatly restricted the application of AMPs.32 Interestingly, the presence of mouse serum only slightly repressed the antimicrobial activity of WRL3 against MRSA. This observation can be explained by an equilibrium between free and protein-bound peptide molecules. As more free molecules become associated with their bacterial targets, the equilibrium would shift toward the progressive release of peptide molecules from serum proteins and contribute to the maintenance of antimicrobial activities.33 The antibacterial activity of WRL3 against MRSA further decreased with an increase in serum

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concentrations. As serum albumin has a negative charge, one explanation for the reduced activity is competition for peptide-binding between serum albumin and bacterial cells. Overall, the great resistance of WRL3 to salts and serum makes it a promising therapeutic for the treatment of bacterial infections. Hemolysis and cytotoxicity, a major bottleneck for the systemic application of AMPs, should be urgently and extensively investigated before expanding the use of AMPs. We found that WRL3 displayed no toxicity toward host cells in vitro. Moreover, AMPs have frequently been assumed to selectively kill bacterial cells without being toxic to host cells.34 We examined the cell selectivity of WRL3 in a co-culture of MRSA and host cells; the results indicated that WRL3 selectively killed bacterial cells and left normal liver cells unharmed. It is possibly due to the fundamental differences between microbial and host membranes that represent potentially selective targets for AMPs. Previous studies have reported that bacterial cytoplasmic membranes composed predominantly of phosphatidylglycerol (PG), cardiolipin (CL), or phosphatidylserine (PS) tend to be highly electronegative. On the contrary, bilayers enriched in the zwitterionic phospholipids, such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), or sphingomyelin (SM), commonly found in mammalian cytoplasmic membranes, are generally neutral in net charge.35-36 Therefore, cationic peptides would preferentially target microbial cells over mammalian cell membranes. These observations give further promise to the use of WRL3 as new antibacterial agent for systemic applications. Unlike conventional antibiotics, such as methicillin and vancomycin, which inhibit specific biosynthetic pathways (e.g., cell wall or protein synthesis), amphipathic AMPs primarily act by

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damaging the bacterial membrane.37 The fluorescence distribution in the bacterial membranes and the model membranes displayed by confocal microscopy provides direct proof that WRL3 targets the bacterial cell membrane. The results of AFM, TEM, flow cytometry, and dye leakage assay confirm that WRL3 induces morphological alterations in the bacterial membrane that leads to the loss of membrane integrity. Upon these results above, we conclude that the initial bactericidal action of WRL3 results from the selective binding to the negatively-charged bacterial cell membrane, followed by damaging the cell membrane integrity via pore formation, leading to the membrane atrophy and fracture, as well as leakage of the intracellular contents. This mechanism of action ensures that WRL3 is a new potential antistaphylococcal drug, with the ability to kill target cells rapidly and the potential to minimize the induction of antibiotic-resistant strains. Bacterial biofilm, a complex bacterial lifestyle adaptation, provides a protection from environmental stresses 5. When biofilms accompany bacterial infections, they make the infections extremely difficult to treat because bacterial cells in a biofilm exhibit stronger resistance to both the host immune system and antibiotic treatments.38 Therefore, once biofilms have colonized the wound, the morbidity and mortality of skin wound patients, as well as treatment cost, will greatly increase, especially, in the case of infections by multiple drug-resistant MRSA. Our data indicate that WRL3 has higher anti-biofilm activities than vancomycin. This superior bactericidal activity against biofilm cells is most likely due to membrane-disruption by WRL3. Mode-of-action studies with living bacterial cells (Figure 5) and artificial membranes (Figure 6) demonstrated that WRL3 initially targeted microbial membranes and subsequently disrupted membrane integrity, eventually led to rapid bacterial cell death (Figure 1). In contrast, vancomycin exhibited a weaker bactericidal activity against biofilm cells. Although the detailed

18


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molecular mechanisms underlying antibiotic resistance of biofilms remain unclear, one of the possibilities is that the extracellular matrix inhibits penetration of antimicrobials.39 Since the division septum is the only site of active cell wall biosynthesis in S. aureus, antibiotics that inhibit the late steps of cell wall synthesis must be able to access lipid II at the septum. Previous studies have demonstrated that vancomycin can penetrate the biofilm matrix and functionally bind to the molecular target lipid II;40 therefore, the lower susceptibility of cells in biofilms to vancomycin may be due to the low growth rate of the biofilm cells. Burn injuries render the host susceptible to bacterial infection because of the large skin defects that are created. Burn-wound infection often causes systemic sepsis and severe septicemia, resulting in an increase in mortality of burn-injured patients. It is estimated that about 75% of mortality following burn injuries are related to infections, such as those caused by Pseudomonas aeruginosa (P. aeruginosa) or MRSA, rather than osmotic shock and/or hypovolemia.41-42 Currently, the antibacterial topical therapies have largely failed to control the high prevalence and progressive nature of burn wound infections. Therefore, an appropriate burn-wound care is important to prevent wound infection and improve patient outcome.43-44 In this study, WRL3 treatment significantly reduced the viable MRSA counts in infected mice, and the reduction in efficiency was greater than that with vancomycin. Gram staining of the infected regions also revealed high levels of MRSA in the infected and methicillin-treated mice, lower levels in vancomycin- and ceftriaxone-treated mice, and almost no MRSA in WRL3 alone and WRL3 + ceftriaxone treated mice. These results indicate that WRL3 is more effective for the treatment of MRSA infection in mice with skin burn wounds. It is likely that the direct bactericidal action of WRL3 is faster than that of antibiotics.

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In addition to control proliferation of MRSA in wound tissue and reduce bioburden, WRL3 also provided a more favorable environment for wound healing, which is a complicated process involving an initial inflammatory response, growth factor production, angiogenesis, tissue cell proliferation and migration, and tissue regeneration and remodeling.45 In this study, WRL3 was found to modulate both immune response and angiogenesis, as evidenced by the inhibition of pro-inflammatory cytokine production and the increase in blood capillary formation, respectively. Furthermore, the results from immunohistochemistry demonstrated that WRL3 treatment effectively increased VEGF production in the infected tissues, which is considered to be a chemoattractant that recruits macrophages and granulocytes, and is involved in nitric oxide-mediated vasodilatation and wound healing through induction of endothelial cell proliferation and blood vessel remodeling.46 We also found that the number of macrophages and monocytes was effectively decreased in WRL3-treated wounds, suggesting that WRL3 can reduce the cytotoxic effects of excessive immune cells. Taken together, these results indicate that WRL3 is most likely involved in antibacterial processes and regulation of tissue homeostasis in the infected wounds, thereby controlling microbial infection and enhancing tissue repair and wound healing (Movie S1 and S2). Thus, the topical application of WRL3-based antimicrobials may provide a novel prophylaxis or treatment strategy for MRSA skin burn wound infections. In conclusion, we demonstrated that an engineered amphipathic peptide, WRL3, displayed potent bactericidal activity against S. aureus, including MRSA, in the absence or presence of physiological concentrations of various salt ions and mouse serum. It also exhibited cell selectivity towards S. aureus cells over host cells in a co-culture model. Additionally, WRL3 was found to effectively disintegrate MRSA preformed biofilms, as evidenced by the substantial reduction in biofilm cell viabilities and

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biomass. Mode-of-action studies with living bacterial cells and artificial membranes reveal that WRL3 selectively binds to the negatively-charged bacterial membrane, damaging membrane integrity and causing intracellular content leakage, eventually leading to cell death. In vivo studies indicated that WRL3 is able to control proliferation of MRSA in wound tissue and reduce bioburden, and provides a more favorable environment for wound healing. Thus, WRL3 represents a promising compound in the development of peptide-based membrane-disruptive antimicrobials with potent antimicrobial activity against MRSA in vitro and high efficacy in treating MRSA burn wound infections in vivo. ■ METHODS For a detailed description of the various methods, see Supporting Information TEXT S1. ■ ASSOCIATED CONTENT Supporting Information TEXT S1: Methods; Supplemental Figure S1: AFM images of MRSA; Supplemental Figure S2: Immunohistochemical staining; Movie S1: MRSA-infected mouse; Movie S2: WRL3-treated mouse. ■ AUTHOR INFORMATION *

Corresponding Author

For Xiaomei Bie, Telephone, +86 25 84396570; E-mail, [email protected]; Postal address: 1 Weigang, Xuanwu District, Nanjing, China. 210095 Author contributions Z. Ma and X. M. Bie conceived and designed this research. J. Z. Han, L. Gao and B. X. Chang performed the experiments. H. Z. Zhao and C. Zhang contributed reagents and interpreted results. Z.

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X. Lu and F. X. LV reviewed and edited the manuscript. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (grant no. 31271828) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). ■ ABBREVIATIONS USED MRSA, methicillin-resistant Staphylococcus aureus; EPS, extracellular polymeric substance; AMPs, antimicrobial peptides; AFM, atomic force microscopy; TEM, transmission electron microscopy; SEM, scanning electron microscopy; PS, phosphatidylserine; GM, geometric mean; LC, lethal concentration; PBS, phosphate-buffered saline; FICI, fractional inhibitory concentration index; MTT, 3-(4, 5-dimethylthiozol-2-yl)-2, 5-diphenyltetrazolium bromide; PI, propidium iodide; FITC, fluorescein isothiocyanate; PG, phosphatidylglycerol; CL, cardiolipin; PC, phosphatidylcholine; CHO, cholesterol; PE, phosphatidylethanolamine; SM, sphingomyelin.

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Table 1. Susceptibility of S. aureus to WRL3 and Conventional Antibiotics. MICsa (µg/mL)

Strains

CROc

METd

VANe

GENf

CIPg

DAP

-

≥64

≥16

≥16

≥16

≥4

-

64

16

1

1

0.5

128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

Lethal concentration (LC) was defined as the lowest concentration of an antimicrobial agent at which

the agent completely killed the bacteria. bMinimal hemolytic concentration (MHC) was determined as the lowest concentration of the antimicrobial that caused 10% hemolysis of erythrocytes. cCytotoxicity was determined as the lowest concentration of the peptide that reduced cell viability of human liver L-O2 cells by 10%.

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Table 3. The Antimicrobial Activities of the Peptide and Antibiotics Alone and in Combination against MRSA MICa Agents

Alone

In combination

FICIb

Interaction

WRL3/antibiotics WRL3

2

/

/

/

Nisin

128

2/64

1.5

Indifference

PLL

4

1/2

1

additivity

CRO

8

0.5/1

0.375

synergy

MET

64

1/8

0.625

additivity

VAN

0.5

1/0.0625

0.625

additivity

GEN

16

1/8

1

additivity

CIP

0.5

2/0.0625

1.125

indifference

a

Minimum inhibitory concentration. bFractional inhibitory concentration index (FICI) for peptide in

combination with antibiotics against MRSA. FICI ≤ 0.5 was considered synergistic, 0.5 < FICI ≤ 1.0 was considered additive, 1.0 < FICI ≤ 4.0 was considered indifferent, and FICI > 4.0 was considered antagonistic.

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Table 4. Effects of Cations and Mouse Inactivated Serum on the Antibacterial Activity of WRL3 and Antibiotics against MRSA. a

MIC (µg/mL) Controlb

NaClb

KClb

NH4Clb

CaCl2b

MgCl2b

ZnCl2b

FeCl3b

25% serum

50% serum

WRL3

2

2

1

4

4

0.5

4

32

8

16

Nisin

128

>256

>256

128

>256

>256

>256

>256

128

>256

PLL

4

32

8

32

8

8

16

64

16

64

CRO

8

16

32

64

32

32

32

32

32

32

MET

64

128

128

128

128

>256

>256

128

256

128

VAN

0.5

1

0.5

0.5

1

0.5

>128

16

1

1

GEN

16

>256

64

64

64

32

64

64

32

64

CIP

0.5

0.25

0.5

0.25

0.25

1

0.5

0.25

0.25

0.25

a

Minimum inhibitory concentrations (MIC) were determined as the lowest concentration of the peptides

that inhibited bacteria growth. bThe final concentrations of NaCl, KCl, NH4Cl, MgCl2, CaCl2, and FeCl3 were 150 mM, 4.5 mM, 6 µM, 1 mM, 2 mM, and 4 µM, respectively, and the control did not contain salt ions or serum.

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Figure 1. Time-kill kinetics of WRL3 and antibiotics against methicillin-resistant S. aureus (MRSA). Dilutions of aliquots taken from 0 to 120 min were plated on MH agar. The graphs were derived from average values of three independent trials.

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Figure 2. Cell selectivity of methicillin, vancomycin and WRL3 in a co-culture model. L-O2 cells (105 cells/well) were infected with methicillin-resistant S. aureus (MRSA, 107 cells/mL) in DMEM medium with no serum or antibiotics. The co-culture was then treated without or with methicillin, vancomycin and WRL3 at 1×MIC. (A) MRSA viability and (B) cell viability after treatment with the agents. The graphs were derived from average values of three independent trials.

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Figure 3. Flow cytometric analysis. Exponential phase methicillin-resistant S. aureus cells were treated with the indicated concentrations of methicillin, vancomycin and WRL3, and cellular fluorescence was analyzed by flow cytometry. The increments of the fluorescence signal represented PI uptake resulting from peptide treatment. (A) No peptide (negative control, 2.4%); (B) methicillin (0.5 × MIC, 3.2%); (C) methicillin (1 × MIC, 3.2%); (D) methicillin (2 × MIC, 4.3%); (E) vancomycin (0.5 × MIC, 5.0%); (F) vancomycin (1 × MIC, 3.9%); (G) vancomycin (2 × MIC, 6.4%); (H) WRL3 (0.5 × MIC, 33.4%); (I) WRL3 (1 × MIC, 67.5%); (J) WRL3 (2 × MIC, 86.7%). 36


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Figure 4. TEM images of MRSA cells treated with peptide and antibiotics at their 1 × MICs. (A) Control; (B) methicillin for 30 min; (C) methicillin for 60 min; (D) vancomycin for 30 min; (E) vancomycin for 60 min; (F) WRL3 for 30 min; (G) WRL3 for 60 min; The control was processed without peptides.

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Figure 5. Confocal microscopic images of MRSA cells treated with FITC-labeled WRL3 at 1 × MIC for (A) 0, (B) 15 min and (C) 30 min. Panels on the left, middle and right represent laser-scanning, merged and transmitted-light scanning images of bacterial cells, respectively.

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Figure 6. Interaction of WRL3 with the negatively charged PG/CL (3:1, w/w) liposomes. After the addition of 2 µl of a 32 µg/mL WRL3, the FITC-labelled peptide started to diffuse into the solution. The images showed that the vesicles were incubated with WRL3 for 0 min (A), 10 min (B), 30 min (C) and 45 min (D).

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Figure 7. The leakage of calcein from the liposomes. The calcein leakage from negatively charged PG/CL (3:1, w/w) liposomes after the addition of the indicated concentrations of (A) WRL3, (B) methicillin or (C) vancomycin. (D) The leakage of calcein from PC/CHO (10:1, w/w) liposomes after the addition of methicillin (MET), vancomycin (VAN) and WRL3 at 8 µg/mL. The arrows indicate the addition of Triton X-100 (0.1%) at 10 min, causing the complete efflux of calcein from the liposomes.

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Figure 8. Anti-biofilm activities of peptide and antibiotics. (A) Cell viability and (B) biomass of methicillin-resistant S. aureus biofilms were decreased after 24 h treatment with various concentrations of methicillin, vancomycin and WRL3 relative to the untreated control. Data shown were means ± SEM of three independent experiments. (C) SEM images of MRSA biofilms treated with the indicated concentrations of WRL3. The control was done without peptides.

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Figure 9. The antimicrobial activity and wound healing activity in vivo of WRL3. A scalded skin region of approximate 1 cm2 on the back of the mice was infected with 50 µL of broth mix containing 108 cfu of MRSA alone, or together with MET, VAN, WRL3, CRO or WRL3 and CRO. (A) The viable bacterial counts in the infected area from the indicated days were cultured and shown as colony forming units (CFU). (B) Wound area at 3, 7, 10, 14 and 17 days after infection. (C) Photographs showing wound regions at 3, 7, 14 and 21 days after MRSA infection. ‘Fatal’ indicate that no mice survived the treatment.

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Figure 10. WRL3 modulated productions of inflammatory cytokines in serum and promoted angiogenesis. A scalded skin region of approximately 1 cm2 on the back of the mice was infected with 50 µL of broth mix containing 108 cfu of MRSA alone, or together with MET, VAN, WRL3, CRO or WRL3 and CRO. (A) Effects of peptide and antibiotics on MRSA-induced IL-6, IL-10, TNF-α and MCP-1 production. (B) At 3 days after inoculation, the infected sections from the indicated treatment groups were subjected to Gram staining. (C) Photographs showing the formation of blood vessels in the burned skin in the indicated treatment groups at 3 (up) and 14 (down) days. Presented figures are representative of three sections examined from 3 mice per group at each time point. 43



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Figure 11. HE photomicrograph of the burned skin tissue. A scalded skin region of approximately 1 cm2 on the back of the mice was infected with 50 µL of broth mix containing 108 cfu of MRSA alone, or together with MET, VAN, WRL3, CRO or WRL3 and CRO. The burned skin area from the indicated treatment groups were fixed and subjected to HE staining at 3, 7, and 14 days post-treatment. The arrows pointed to the hair follicles, the arrowheads pointed to epidermal layers, the circles pointed to blood vessels. When compared with vancomycin, more new blood vessels were observed in the injured area treated with WRL3 or WRL3+CRO at 3 and 7 days post-infection. Presented figures are representative of three sections examined from 3 mice per group at each time point. 44


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Membrane-Active Amphipathic Peptide WRL3 with ... - ACS Publications

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