Communication pubs.acs.org/bc
In Vivo Detoxification of Lipopolysaccharide by Antimicrobial Peptides Wenxu Zhang,† Jiangcheng He,† Junchen Wu,*,† and Carsten Schmuck‡ †
Key Laboratory for Advanced Materials & Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China ‡ Institute for Organic Chemistry University of Duisburg-Essen, 47057 Essen, Germany S Supporting Information *
ABSTRACT: Abundant lipopolysaccharide (LPS) can result in sepsis and septic shock, indicating a serious Gram-negative bacterial contamination. We have developed a novel strategy based on dendritic antimicrobial peptides that can detoxify LPS. The dendritic antimicrobial peptides bind to LPS at the surface of Gram-negative bacteria, killing the bacteria but removing the LPS from the cell wall of dead Gram-negative bacteria, hence detoxifying pathogenic bacteria in its host cells and effectively improving survival of animals infected with Pseudomonas aeruginosa. Our findings provide a way to detoxify bacterial contamination.
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INTRODUCTION Lipopolysaccharide (LPS), also known as bacterial endotoxin, is the main structural component of the outer cell membranes of all Gram-negative bacteria.1,2 It is responsible for the integrity and low permeability of the membrane, thereby protecting bacteria from attack by antibiotics.3 Abundant LPS is an indication of bacterial contamination and can result in serious sepsis and septic shock,4 which caused approximately 150 000 casualties annually in the USA.5 The fact that LPS and its resultant macrophage cytokines are primary effectors in the etiology of sepsis strongly suggests that binding to and consequently neutralizing LPS may be of potential therapeutic benefit in septic patients.6−8 Thus, the development of specific antibiotics that can be used to detoxify LPS has become a challenging field of current research.9−11 In recent years, antimicrobial peptides (AMPs) have increasingly been a center of attention because of their multiple modes of action for binding and inactivating pathogens by targeting the bacterial cell membrane, in particular, binding to LPS at the surface of Gram-negative bacteria.12−14 Cationic AMPs serve as the first line of host defense against bacteria, fungi, and viruses, and offer an opportunity to detoxify LPS contamination in Gram-negative bacterial.15 The large number of anionic LPS layer on the outer leaflet of the out membrane of Gram-negative bacteria could be disrupted by AMPs.16 Also, Anirban Bhunia et al. had developed antimicrobial peptide to kill bacterial both in mammalian cells and plants.8,17 Although AMPs mimics have been widely studied to develop new antibiotics,18 they are much less used to detoxify LPS. In this context, a naturally magainin containing 23 amino acids was used as our study model.19,20 This peptide, which has two forms with slightly different sequences [magainin-I (MGN-I) © XXXX American Chemical Society
and magainin-II (MGN-II)], was originally isolated from the skin of the African clawed frog and exhibits a wide range of antimicrobial activities.21 The outer monolayer of the outer membrane in the Gram-negative bacteria is composed of lipid A (Figure 1a). Lipid A, a part of LPS, is a specific phospholipid and served as the main toxic determinant of LPS.2 Its pharmacological activity, particularly on the immune system of animal cells, is considerable. Searching for a suitable antibiotic peptides based on the dual action of the detoxification of LPS, we were drawn to the combination of polymyxin B (PMB) topology and magainin sequence. Thus, the newly designed peptides containing a naphthalimide fluorophore and four β-alanines differ from maganins in structures. Introduced naphthalimide fluorophores can be utilized to track the interaction between peptides and LPS.22 Subsequently, the tailor-made peptides were successfully synthesized by solid-phase peptide protocol and purified to 99.0% (M-MGN-I) and 99.2% (M-MGN-II) purity with yields of 5.4% (M-MGN-I) and 3.2% (M-MGN-II) after cleavage and deprotection (Figure 1c). In addition, MGNs were synthesized and used as a control (for details, see Supporting Information Tables S1−S4).23
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RESULTS AND DISCUSSION To gain insight into the interaction between the peptides and LPS, fluorescent spectra were used to monitor any binding induced changes. Upon the addition of LPS, the fluorescence Received: November 17, 2016 Revised: December 16, 2016
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DOI: 10.1021/acs.bioconjchem.6b00664 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Communication
Bioconjugate Chemistry
Next, circular dichroism (CD) spectra were used to evaluate the conformation of M-MGNs in the presence and absence of LPS (Figure S3a,b).The CD spectra of M-MGNs exhibited a weak negative Cotton effect at 225 nm, whereas after adding LPS, they displayed strong peaks at 225 nm. This result indicated that binding to LPS induced α-helix formation, which were identical with a previous report.28,29 The ability of AMPs to perturb the micelle structure of LPS has been correlated with their antimicrobial and anti-endotoxic activities.30 Therefore, dynamic light scattering (DLS) was carried out to determine the structural perturbation of LPS lipids by M-MGNs. Figure S3 shows DLS measurements of LPS aggregate size in the absence and presence of M-MGNs. In MGN-free solution, LPS micelles assembled into larger aggregates with sizes from 92 to 5533 nm (Figure S4a). Similarly, M-MGN-I can form structures with hydrodynamic diameters ranging from 0.6 to 5509 nm (Figure S4b). Once M-MGN-I bound to LPS, smaller clusters of LPS aggregates were observed with hydrodynamic diameters from 56 to 488 nm (Figure S4c). The same phenomena were observed with M-MGN-II (Figure S4d,e). Hence, the M-MGNs interfered with the aggregation of LPS, which could improve their anti-endotoxic activity. Next, to determine the contribution of the phosphate anion of LPS and the positive charge of the M-MGNs, the thermodynamic parameters for the binding of LPS and peptides were measured using an isothermal titration calorimeter (ITC) (Figure S5a,b and Table S5). The binding of M-MGNs to LPS was endothermic and was mainly driven by entropy. The binding constants were also rather low (Ka = 105−106 M−1), indicating that the binding of M-MGNs to LPS occurs mainly because of nonspecific charge interactions and the displacement of multiple associated monocations from the LPS upon the binding of a single polycationic M-MGNs.31 The efficacy of M-MGNs in killing clinically significant pathogens was tested using six Gram-negative strains (Escherichia coli ATCC-25922, Klebsiella pneumonia ATCC700603, Pseudomonas aeruginosa ATCC-27853, Shigella dysenteriae ATCC-13313, Acinetobacter ATCC-19606, and Citrobacter freundii ATCC-13316). Compared with MGNs, MMGNs exhibited a marked decrease in the minimum inhibitory concentration values against these Gram-negative bacteria (Figure S6−S7).32,33 These results are consistent with half maximal inhibitory concentration (MIC50) (Figure 3). In particular, for P. aeruginosa, the MICs of M-MGN-II (13.9 μg/ mL) and M-MGN-I (13.1 μg/mL) were significantly lower than those of MGN-II (69.2 μg/mL) and MGN-I (118 μg/ mL). To test the effect of M-MGNs on the membrane integrity of P. aeruginosa, β-galactosidase leakage was assessed (Figure S8a,b). MGNs and M-MGNs showed no β-galactosidase leakage at their MICs or even at concentrations of up to 5fold their MICs. These data support a mechanism of antibacterial action mainly attributable to penetration of peptide to the bacterial membrane. To determine the effect of M-MGNs on the cell membrane, CLSM was used to confirm whether the cultures were dead or alive. In a colocalization experiment, P. aeruginosa was incubated with M-MGNs (at the MIC and 5-folds MIC) for 1.0 h, and propidium iodide (PI) staining was performed to discriminate viable from nonviable P. aeruginosa upon CSLM observations; green and red signals in the P. aeruginosa images were from M-MGNs and PI (Figure 2), respectively. The overlay images demonstrate that MMGNs caused bacterial death after penetrating the bacterial membrane. We can clearly see that the P. aeruginosa treated
Figure 1. (a) Structures of Lipid A; (b) Schematics of MGNs and MMGNs synthesized by the solid-phase method.
emission of M-MGNs containing a naphthalimide fluorophore increased markedly and showed a blue-shift from 550 to 525 nm. These changes in fluorescence were most likely due to the increased hydrophobic microenvironment around the naphthalimide fluorophore.24,25 The hydrophobic microenvironment provided by the LPS enhances the emission of naphthalimide fluorophores in aqueous solution (Figure S1a,b). The changes in fluorescence clearly demonstrated that M-MGNs bind effectively to LPS. Furthermore, confocal laser scanning microscopy (CLSM) was used to confirm whether MMGNs were encapsulated in LPS micelles.26 As seen in Figure S2a, intense green luminescence was detected when the molar ratio of LPS/M-MGN-I reached 2. In Figure S2b,d, we can clearly see that the gray area around the green fluorescence MMGNs denotes the LPS micelles. Moreover, the overlay image revealed that the M-MGN-I was encapsulated in LPS micelles (Figure S2b,c), which is in good agreement with literature.27 Similarly, the results for M-MGN-II are shown in Figure S2d−f. B
DOI: 10.1021/acs.bioconjchem.6b00664 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Communication
Bioconjugate Chemistry
Figure 2. CLSM images of P. aeruginosa incubated with 13.1 μg/mL (MIC) or 65.5 μg/mL (5-fold MIC) M-MGN-I or13.9 μg/mL (MIC) or 69.5 μg/mL (5-fold MIC) M-MGN-II at 37 °C for 1 h and then stained with 2 μg/mL PI for 30 min in TBS solution (pH 7.4, 50 mM Tris, 10 mM NaCl, 25 °C). P. aeruginosa treated with M-MGNs at 5-fold MIC shows much more death-staining than that at MIC (Channel 1: excitation: 405 nm, emission collected: 533−573 nm; Channel 2: excitation: 515 nm, emission collected: 593−643 nm. Scale bar, 5 μm).
concentration of amphipathic α-helix conformation in the LPS containing bacterial membrane. The biocompatibility and in vitro stability of MGNs and MMGNs were used to obtain the concentrations required for the detoxification, and these experiments were evaluated by cytotoxicity, hemolytic activity, and trypsin degradation assays. First, the cytotoxicity of MGNs and M-MGNs was measured using MTT assays in human nonsmall cell lung cancer A549 cells, human bronchial epithelial (HBE) cells, human oral epidermoid carcinoma (KB) cells, and HeLa cells (Figure S9). At MGNs and M-MGNs concentrations
