Self-Assembling Myristoylated Human Î±-Defensin 5 as a Next

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Self-Assembling Myristoylated Human #-Defensin 5 as a Next-Generation Nanobiotics Potentiates Therapeutic Efficacy in Bacterial Infection ruyi lei, Jinchao Hou, QiXing Chen, Weirong Yuan, Baoli Cheng, Yaqi Sun, Yue Jin, Lujie Ge, Shmuel A. Ben-Sasson, Jiong Chen, Hangxiang Wang, Wuyuan Lu, and Xiangming Fang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b09109 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Self-Assembling Myristoylated Human α-Defensin 5 as a Next-Generation Nanobiotics Potentiates Therapeutic Efficacy in Bacterial Infection Ruyi Lei1, #, Jinchao Hou1, #, Qixing Chen2, Weirong Yuan3, Baoli Cheng1, Yaqi Sun1, Yue Jin1, Lujie Ge1, Shmuel A. Ben-Sasson4, Jiong Chen5, Hangxiang Wang6, *, Wuyuan Lu3, *, Xiangming Fang1, *

1, Department of Anesthesiology and Intensive Care, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China 2, The Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310052, China 3, Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA 4, Department of Developmental Biology, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 5, Laboratory of Biochemistry and Molecular Biology, Ningbo University, Ningbo 315211, China 6, Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China

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ABSTRACT: The increasing prevalence of antibacterial resistance globally underscores the urgent need to the update of antibiotics. Here, we describe a strategy for inducing the self-assembly of a host-defense antimicrobial peptide (AMP) into nanoparticle antibiotics (termed nanobiotics) with significantly improved pharmacological properties. Our strategy involves the myristoylation of human alpha-defensin 5 (HD5) as a therapeutic target and subsequent self-assembly in aqueous media in the absence of exogenous excipients. Compared with its parent HD5, the C-terminally myristoylated HD5 (HD5-myr)-assembled nanobiotic exhibited significantly enhanced broad-spectrum bactericidal activity in vitro. Mechanistically, it selectively killed Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) through disruption of the cell wall and/or membrane structure. The in vivo results further demonstrated that the HD5-myr nanobiotic protected against skin infection by MRSA and rescued mice from E. coli-induced sepsis by lowering the systemic bacterial burden and alleviating organ damage. The self-assembled HD5-myr nanobiotic also showed negligible hemolytic activity and substantially low toxicity in animals. Our findings validate this design rationale as a simple yet versatile strategy for generating AMP-derived nanobiotics with excellent in vivo tolerability. This advancement will likely have a broad impact on antibiotic discovery and development efforts aimed at combating antibacterial resistance.

KEYWORDS: self-assembly, antimicrobial peptide, sepsis, infection, antibiotic resistance

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Incidents of infection caused by antibiotic-resistant bacteria have been steadily increasing globally and are predicted to kill 10 million people annually by 2050.1 An important compounding factor in this predicament is the lack of the update on antibiotics for the past several decades, as most antibiotics in current use were derived from natural microbial products discovered from the 1940s to the 1960s.2 Thus, the anti-infective therapeutics refractory to existing resistance mechanisms are urgently needed to combat the increasing prevalence of antibacterial resistance. Since de novo antibiotic drug discovery based on the chemical entities is challenging, the exploitation of natural products such as host-defense factors as a valuable source of antimicrobials may be an attractive approach to the development of next-generation antibiotics.2-4

Mammalian antimicrobial peptides (AMPs) are an important class of host-defense factors with broad antibacterial, antiviral and antifungal activity.4 Defensins of the alpha and beta families, which are mainly expressed in phagocytes and epithelial cells, constitute the main class of AMPs in humans.5-7 Most AMPs are thought to kill bacteria through membrane disruption, a process that entails transmembrane pore formation, subsequent cytoplasmic content leakage of

and

ultimately cell death.8-9 More recent work has shown that AMPs such as human alpha-defensins and beta-defensin 3 also kill Gram-positive bacteria by inhibiting bacterial cell wall synthesis.10-11 These modes of antimicrobial action likely endow AMPs with the ability to escape some known mechanisms of antibacterial resistance,12 making them attractive scaffolds for the creation of therapeutics superior to traditional antibiotics.

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However, naturally occurring AMPs generally exhibit only modest therapeutic efficacy and are prone to losing potency under physiological conditions after systemic administration. This phenomenon occurs because AMPs are susceptible to proteolytic degradation in vivo and because their bactericidal activity can be negated in body fluids by salts and anionic substances (proteins, nucleic acids, polysaccharides, etc.) that are known to interfere with the action of AMPs through nonspecific electrostatic interactions.13-14 Thus, harnessing AMPs as a class of antibiotics necessitates the development of therapeutically viable strategies to overcome these pharmacological limitations, i.e., their low bioavailability and poor in vivo stability.

Noncovalent self-assembly of individual molecular building blocks has provided innovative approaches to the creation of fascinating nanometer-scale structures for extensive biomedical application.15-17 For example, Liu et al. recently demonstrated that an amphiphilic construct, comprising an N-terminal (hydrophobic) cholesterol moiety, an Arg-rich antimicrobial peptide of six amino acid residues and a C-terminal cationic cell-penetrating peptide, self-assembled into positively charged core-shell micelles in solution.18 These nanoparticulate antibiotics showed a high therapeutic index against Staphylococcus aureus (S. aureus) infection in mice and rabbits.18-19 Inspired by these findings, we here present a simple yet versatile strategy that combines structurally rational reconstitution of an AMP with subsequent supramolecular nanoassembly to construct a kind of nanotherapeutics with significant potential in the treatment of a broad range of bacterial infections.

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Myristoylated HD5 self-assembles into supramolecular nanostructures in solution. To validate this strategy, we chose human alpha-defensin 5 (HD5) as a target AMP. HD5 possesses 32 amino acid residues and is expressed in abundance in the small intestine by Paneth cells, which play critical roles in the innate host defense against various enteropathogens.20-21. HD5 adopts a three-stranded beta-sheet structure stabilized by three intra-molecular disulfides bonds.22 While hydrophobicity and selective cationicity play critical roles in HD5 function,23-24 this defensin is also highly water soluble and likely exists as a dimer that functions under physiological conditions.25 To impart more hydrophobicity to HD5 for facilitating its nanoassembly in aqueous solutions, we coupled myristic acid, a 14-carbon saturated fatty acid, to its N- or C-terminus via an amide bond (Figure 1A). For myristoylation, HD5 was chemically synthesized via solid-phase peptide synthesis (SPSS)26 and extended N-terminally by a Gly residue or C-terminally by a Gly-Lys linker (Figure 1B), resulting in two myristoylated forms of HD5, hereinafter referred to as myr-HD5 and HD5-myr, respectively. Oxidative folding of HD5, myr-HD5 and HD5-myr in the presence of oxidized and reduced glutathione was essentially as described.27 All peptides were purified to >95% purity by high-performance liquid chromatography (HPLC) and verified by electrospray ionization mass spectrometry (ESI-MS) (Figure S1-S3). Circular dichroism (CD) analysis has been routinely performed to estimate the secondary structure of proteins because of the fingerprint signatures of α and β domains.28 The CD spectra of myristoylated HD5 exhibited a negative band in the far-UV region at 209 nm typical of a β-sheet structure (Figure S4 and Table S1). Both of these CD spectra are consistent with that of native HD5, confirming their correct

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folding into β-sheets and the formation of disulfide bonds even after the modification with myristic acid.

With these two HD5 derivatives in hand, we next used transmission electron microscopy (TEM) to investigate their self-assembly behavior by preparing aqueous suspensions of both myristoylated constructs. Intriguingly, blending myr-HD5 and HD5-myr with deionized (DI) water at a final concentration of 200 µg/ml resulted in the spontaneous formation of sub-50 nm spherical structures (Figure 1C). Further particle size analysis by dynamic light scattering (DLS) revealed average hydrodynamic diameter (dh) values of 80.2±2.2 and 56.0±8.4 nm for myr-HD5 and HD5-myr, respectively, with a relatively narrow polydispersity index (Figure 1D). However, their unmodified counterpart, HD5, did not show any nanoassembly formation under TEM observation. The stability of the HD5-myr nanobiotic was further examined in different aqueous media such as water, sodium chloride (NaCl), DMEM and mouse serum. As shown in Figure S5-8, native HD5 was found to undergo substantial hydrolysis in serum within 24 h, whereas HD5-myr remained stable in all media during the incubation time. Thus, HD5-myr was resistant to hydrolysis by proteolytic enzymes due to the self-assembled nanostructures. Together, these results unequivocally confirm that myristoylated HD5 derivatives can form systemically injectable and stable nanoassemblies without any exogenous excipients, thereby supporting our design rationale for generating HD5-derived nanobiotics through simple peptide myristoylation.

C-terminally myristoylated HD5 potently kills a broad range of bacteria in vitro. We used the previously published virtual colony-count assay to quantify the antimicrobial activity of

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HD5,20 the myr-HD5 nanobiotic and the HD5-myr nanobiotic against an array of Gram-positive and Gram-negative bacteria, including some clinical strains frequently encountered in patients.1, 29 Following treatment with different concentrations of peptides for 2 h, bacterial cells (2×106 CFU/ml) were added to an equal volume of double strength (2×) LB and their growth kinetics were continuously monitored at 37 °C on a 96-well plate reader to determine bacterial survival. As shown in Figure 2A-F, while HD5 had negligible bactericidal activity against S. aureus and methicillin-resistant Staphylococcus aureus (MRSA) at concentrations up to 12.5 µg/ml, it only weakly inhibited the growth of E. coli, Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) under the same conditions. Similar to HD5, the myr-HD5 nanobiotic was generally ineffective in killing bacterial except S. aureus and MRSA: the myr-HD5 nanobiotic at 12.5 µg/ml reduced the survival of both strains by approximately four orders of magnitude (Figure 2A-B). By contrast, the HD5-myr nanobiotic was substantially more potent than either HD5 or the myr-HD5 nanobiotic against all six strains tested. While the HD5-myr nanobiotic at 6.25 µg/ml reduced the survival of S. aureus by four orders of magnitude, it killed more than 99.9999% (a 6-log reduction in bacterial survival) of MRSA, E. coli, A. baumannii, P. aeruginosa and K. pneumoniae at 12.5 µg/ml. In the presence of NaCl, the bactericidal activity of HD5 was negligible, which was in accordance with a previous report.30 However, the HD5-myr nanobiotic could considerably reduce the survival of E.coli at 25 µg/ml even at high concentration of NaCl (e.g., 150 mM, Figure S9). The presence of high concentrations of NaCl increased the dh values of self-assembled HD5-myr particles (Figure S10) but did not decrease their bactericidal potency. More importantly, the bactericidal activity of the 7


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HD5-myr nanobiotic at a concentration of 50 µg/ml was largely maintained when tested against E. coli and MRSA in the presence of 50% mouse serum (Figure 2G-H). Overall, the in vitro antimicrobial activity data indicate that the C-terminally myristoylated defensin HD5-myr nanobiotic is a broad-spectrum nanobiotic and superior in potency to HD5 and its N-terminally myristoylated counterpart. Our subsequent efficacy and mechanistic studies were therefore focused primarily on the HD5-myr nanobiotic.

HD5-myr nanobiotic rescue mice from lethal sepsis induced by systemic E. coli infection. Encouraged by the favorable in vitro activity profile of the HD5-myr nanobiotic, we evaluated its therapeutic potential using an in vivo animal model where mice (in 4 groups of 10 mice each) were infected with E. coli injected intraperitoneally at a lethal dose of 7×106 CFU per mouse.31 Immediately after infection, the three treatment groups received a single dose of HD5 at 10 µg per mouse, the HD5-myr nanobiotic at 10 µg per mouse, or the HD5-myr nanobiotic at 20 µg per mouse, whereas the control group was injected with a vehicle. As shown in Figure 3A, without treatment, none of the ten mice survived beyond 24 h postinfection. Although HD5 treatment improved the survival to 30% at 48 h, no statistically significant difference from the vehicle-treated control group was observed. By contrast, HD5-myr nanobiotic treatment significantly prolonged the survival of infected mice in a dose-dependent manner. While seven of ten mice in the low-dose group were alive at 48 h, nine of them in the high-dose group were rescued by the HD5-myr nanobiotic (Figure 3A).

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Severe bacterial infection in animals causes loss of body weight, which itself is an important parameter for therapeutic efficacy.32-34 We monitored the body weight of the ten surviving animals (3 from the HD5-treated group and 7 from the low-dose HD5-myr nanobiotic-treated group) throughout the experiment for up to six days postinfection. As shown in Figure 3B, mice treated with the HD5-myr nanobiotic recovered their body weight more quickly and robustly than did their HD5-treated counterparts. In fact, while a significant loss of 17.8 ± 2.9% in average body weight was observed at 48 h post infection for HD5-treated mice, the body weight loss in HD5-myr nanobiotic-treated mice was only 8.2 ± 4.0% (Figure 3C).

Acute organ injury and failure are hallmarks of septic shock, as manifested in murine models by sepsis-associated severe inflammatory infiltrates in the lung and liver.35-37 We examined these organs harvested from normal mice and from the three groups of E. coli-challenged mice 8 h after treatment with vehicle, HD5 (10 µg per mouse) or the HD5-myr nanobiotic (10 µg per mouse). As shown in Figure 3D, overt inflammation of injured organs was evident in vehicle - or HD5-treated mice. However, similar to those of the normal mice, the lung and liver of HD5-myr nanobiotic-treated mice appeared comparable (Figure 3D). Taken together, these in vivo data obtained from the murine sepsis model demonstrate the superior therapeutic efficacy of the HD5-myr nanobiotic in systemic bacterial infections.

HD5-myr nanobiotic protects against sepsis in mice by reducing bacterial burden and organ injury. To obtain insight into the mechanisms by which the HD5-myr nanobiotic effectively protects mice from infection, we assessed its bactericidal effect in vivo. Accordingly, mice (n = 10

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per group) were infected with a lethal dose of E. coli (7×106 CFU per mouse), and then administered a single-dose treatment of vehicle or 20 µg of HD5 or the HD5-myr nanobiotic per mouse. At 8 h posttreatment, samples were collected from harvested organs, blood and peritoneal lavage fluids (PLFs) for quantification of E. coli using a colony-count assay. As shown in Figure 4, both HD5 and the HD5-myr nanobiotic reduced the bacterial burden in the liver, lung, spleen, kidney, blood, and PLF of the infected mice. However, the HD5-myr nanobiotic did so much more effectively than HD5. High bacterial burden in mice is predictably associated with exacerbated organ injury.38 As shown in Figure 4B, the lung of vehicle-treated mice displayed large amounts of effusion in the alveolus and thickened respiratory membranes; a large thrombus formed in the liver accompanied by congestion and expansion of the hepatic sinusoid; and many casts formed in the renal tubules and capsules in the kidney. In contrast, HD5 treatment partially alleviated these abnormalities associated with sepsis-induced tissue damage, whereas treatment with the HD5-myr nanobiotic largely eliminated them (Figure 5B). Organ injury score analysis (Figure 4C-E) fully corroborated these findings, underscoring the ability of the HD5-myr nanobiotic to protect mice from sepsis by reducing bacterial burden and alleviating organ injury.

HD5-myr nanobiotic protect mice against skin infection by MRSA. S. aureus, particularly rapidly emerging strains of MRSA, is a major pathogen associated with serious community-acquired and nosocomial diseases, necessitating the development of antibiotics to treat S. aureus infections. We further evaluated the therapeutic efficacy of the HD5-myr

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nanobiotic in a mouse model of bacterial skin infection by a MRSA strain, ATCC 43300, that replicates typical S. aureus infections in humans.39-40 Shaved mouse skin was tape-stripped to disrupt the skin barrier and infected by epicutaneously inoculated MRSA on filter paper discs as previously described (Figure S11).41-43 At 24 h after the infection had been established, sterile filter paper discs containing the HD5-myr nanobiotic (12 µg) and vehicle were patched onto the sites of infection for another 24 h, followed by quantification of the colonized MRSA on the skin and in subcutaneous tissues. As shown in Figure 5A, compared with the HD5-myr nanobiotic-treated mouse skin, the vehicle-treated skin was substantially more inflamed, as evidenced by the accumulation of large amounts of pus. As expected, treatment with the HD5-myr nanobiotic led to a significant reduction in the colonization of MRSA both on the epidermis and in subcutaneous tissues (Figure 5B-C). By contrast, treatment with HD5 was ineffective (Figure 5D-E). These data suggest that the HD5-myr nanobiotic can penetrate through the skin into diseased sites to eradicate MRSA, indicating its promise as part of a class of antibiotics with significant therapeutic potential for external use.

HD5-myr nanobiotic is not toxic in vitro and in vivo. Side effects caused by AMPs impede their clinical use as promising therapeutics. Prior studies indicated that some cationic peptides could interact with the cell membrane of red blood cells (RBC) and exert significant hemolytic activity.3 To better understand the toxicity of the HD5 nanobiotics, we evaluated the hemolytic activity of HD5, the myr-HD5 nanobiotic and the HD5-myr nanobiotic in vitro using a previously described method.18 Liposomal amphotericin B, an effective antibiotic widely used in patients,44-45 was used as a control. As shown in Figure S12, neither HD5 nor the HD5 nanobiotics exhibited meaningful 11


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hemolytic activity even at relatively high concentrations. This result is in stark contrast to the finding obtained with liposomal amphotericin B, which caused approximately 34% hemolysis at 400 µg/ml. Interestingly, the HD5-myr nanobiotic was noticeably less hemolytic than its N-terminally myristoylated counterpart. In addition, a mouse macrophage cell line, namely, RAW 264.7 was chosen to evaluate the cytotoxicity induced by the HD5-myr nanobiotic using the MTT assay. Almost no apoptotic cell was observed in the presence of HD5-myr at 100 µg/ml; however, treatment with liposomal amphotericin B at 100 µg/ml resulted in a cell viability of only 40% (Figure S13). To further evaluate the safety of the HD5-myr nanobiotic in vivo, we injected mice (n=5) intraperitoneally with the HD5-myr nanobiotic (60 µg per mouse) and monitored the change in body weight for seven days. As shown in Figure S14, all five mice remained healthy as indicated by the gradual gain in body weight observed over this time period, confirming a favorable toxicity/safety profile of the HD5-myr nanobiotic in vitro and in vivo.

HD5-myr nanobiotic most likely kills bacteria through disruption of the bacterial membrane and/or cell wall structure. The complex mechanism by which AMPs kill bacteria is well documented in the literature.9 For native HD5, several studies disclosed the direct attack of bacteria by HD5.46 To visualize the interaction between the HD5-myr nanobiotic and bacteria, we also

synthesized

TAMRA-labeled

HD5

and

HD5-myr

(termed

TAMRA-HD5

and

TAMRA-HD5-myr, respectively) and subjected then to a confocal microscopy-based study (Figure S15). The addition of TAMRA-HD5 to EGFP-E. coli (E. coli expressing enhanced green fluorescence protein) led to rapid cellular uptake within several minutes; however, TAMRA-HD5-myr showed a higher binding and penetrating activity than TAMRA-HD5 (Figure 12


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S16). To better understand the modes of action of the HD5-myr nanobiotic against bacteria, we used scanning electron microscopy (SEM) to investigate the morphological changes in two representative bacterial strains, MRSA and E. coli, in response to peptide treatment. As shown in Figure 6, treatment of MRSA and E. coli with either HD5 or myr-HD5 nanobiotic at a concentration of 100 µg/ml for 2 h led to the formation of blebs (roughness) on the bacterial surface. In contrast, treatment of E. coli with the HD5-myr nanobiotic created irregularly shaped holes in the membrane, whereas the C-terminally myristoylated defensin caused rupture of the MRSA bacterial membrane. Thus, the antimicrobial activity of HD5, myr-HD5 nanobiotic and the HD5-myr nanobiotic appears to be positively correlated with their ability to induce morphological changes in both E. coli and MRSA. Considering the known mechanisms of the antibacterial activity of human alpha-defensins,9, 46 it is conceivable that disruption of the bacterial membrane and/or cell wall structure, as evidenced by the SEM data, is the cause of lethality in bacteria treatment with the HD5-myr nanobiotic.

To further explore whether the inhibition of bacteria was a consequence of the HD5-myr nanobiotic targeting in vivo, we used two-photon laser scanning microscopy (TPLSM) to get a visual interpretation in vivo. Sepsis was induced by intravenous injection of the EGFP-E. coli immediately followed by injection of TAMRA-HD5, TAMRA-HD5-myr or PBS. After 30 min, the mesenteric vein was exposed and observed by two-photon confocal microscopy. The flow of EGFP- E. coli in the blood vessels could be clearly observed (Figure S17, Video S1-3). We interestingly found that both HD5 and the HD5-myr nanobiotic could bind with EGFP- E. coli in the blood at 30 min after sepsis inset. Moreover, the number of EGFP-E. coli cells in the blood 13


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stream of TAMRA-HD5 and PBS-treated mice was comparable. However, the EGFP- E. coli number was significantly decreased in the TAMRA-HD5-myr-treated mice.

DISCUSSION Antibiotic resistance is a natural response of bacteria evolving and adapting under constant selective pressure. Bacteria evade antibiotics using many different mechanisms, including altering their membrane permeability to control the uptake or efflux of molecules, enzymatically inactivating antibiotics, and modifying the intended target of drug intervention. Incidents of antibiotic resistance are emerging at an alarming rate in hospitals and communities globally as existing antibiotics continue to lose their effectiveness. This dire situation has been aggravated by, among many other factors, excessive and inappropriate uses of antibiotics in agriculture and human medicine as well as steadily declining efforts in antibiotic discovery and development by the pharmaceutical industry.

It has been more than 30 years since the last introduction of a class of antibiotics, i.e., daptomycin – a naturally occurring lipopeptide from a microbial source that kills drug-resistant bacteria by disrupting multiple aspects of bacterial cell membrane function.47 The distinct mechanism of action of daptomycin is reminiscent of the antibacterial mode of action of mammalian AMPs such as defensins.48 As is the case with AMPs, which largely target the bacterial cell membrane and/or cell wall synthesis machinery,9, 49 daptomycin rarely elicits the drug resistance observed with traditional antibiotics.47 Not surprisingly, much effort has been made in the past several decades to develop AMPs as anti-infective therapeutics for clinical use.3 However, little success has been 14


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achieved due to the inherent pharmacological limitations of small peptides in general. Considering the discovery of daptomycin and recent advances in nanobiotics,18-19,

50

it is

conceivable that a lipidated host-defense peptide would form nanobiotics with improved pharmacological properties,28,

51-54

thereby promising a therapeutically viable strategy for the

update of antibiotics against drug-resistant bacteria. Our data on the self-assembled HD5-myr nanobiotic clearly validates this general design strategy and provides a conceptual framework for AMP-based antibiotic drug discovery aimed at combating antibacterial resistance.55

In this report, we have shown that C-terminal myristoylation enables HD5 to readily form supramolecular nanoassemblies independent of exogenous matrices. Unexpectedly, the self-assembly of HD5-myr nanoparticles translates this important host-defense peptide into a nanobiotics with significantly improved pharmacological properties. HD5 itself effectively kills both Gram-positive and Gram-negative strains of bacteria in vitro, presumably through disruption of the bacterial cell membrane and/or cell wall synthesis machinery.25, 46 However, the presence of physiologically relevant components substantially reduces the antimicrobial activity of HD5, thereby impeding its clinical translation. Compared with unmodified HD5, the HD5-myr nanobiotic exhibited potent bactericidal activity with broad specificity and displayed impressive therapeutic efficacy in bacterial infection by reducing systemic bacterial burden and inflammatory organ damage in mice. In addition, the HD5-myr nanobiotic showed a favorable safety profile in vivo, indicating its promise as part of HD5-derived anti-infective agents with significant therapeutic potential in the treatment of human infections. Similar to many other AMPs, whose target is the bacterial outer membrane,56 HD5 also could not cause damage to the inner membrane 15


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of Gram-negative bacteria.28 However, the HD5-myr nanobiotic seems to possess the ability to disrupt the whole cell membrane and cell wall of E.coli as evidenced by the results of the SEM-based studies shown in Figure 6. This distinct bactericidal mechanism makes HD5-myr nanobiotics a promising candidate to kill bacteria strains that are resistant to traditional antibiotics. Moreover, the disruption of the whole bacterial membrane upon HD5-myr treatment may facilitate the entry of other antibiotic, which implies the potential use in drug combination therapy.

While cationicity plays an important role in the killing of Gram-negative bacteria by human alpha-defensins,4 hydrophobicity is critical for killing Gram-positive strains.23 Defensins clustered in nanoassemblies necessarily carry a significantly higher density of charged and hydrophobic residues, thereby contributing to the more effective and efficient killing of bacteria by the HD5-myr nanobiotic than by its parent HD5. The fact that the HD5-myr nanobiotic, assembled into higher-order structures, is more active in bacterial killing than its parent HD5, at least partially supports the previous finding that HD5 dimerization is essential for its antibacterial function.23 Furthermore, the HD5-myr nanobiotic is more resistant to proteolytic degradation in vivo than its unmodified counterpart as clustered defensin molecules collectively create steric hindrance to shield one another from hydrolysis by proteolytic enzymes.57-58 In addition to the enhanced in vivo stability of the HD5-myr nanobiotic, nanoparticle formation increases the apparent molecular size to such an extent that renal excretion is likely minimized, possibly improving the bioavailability of the HD5-myr nanobiotic. Unexpectedly, although N-terminal myristoylation endowed the defensin peptide with the ability to form nanoparticles in aqueous 16


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solution and showed the improved its bactericidal activity against Gram-positive bacteria, the potency

in

killing

Gram-negative

bacteria

strains

was

significantly

reduced.

This

bacteria-selective activity implies that HD5 may kill different bacteria through different mechanisms and that some specific structures may be important for its activity against specific bacteria. It has been found that the active region of HD5 is located at the C-terminus. We hypothesize that once the nanoparticles are embedded in the bacterial cell membrane, they interact with the cell membrane components on the surface of the bacteria. When the aqueous environment is replaced by the phospholipid bilayer environment, the nanoparticles may disassemble. Then, the monomers continue to interact with the cell membrane and exert a bactericidal effect. We speculated that modification of different terminuses in HD5 may affect the bacterial selectivity of the resultant monomers, resulting in the different bactericidal activities between myr-HD5 and HD5-myr.

CONCLUSIONS AND PROSPECTS In summary, by exploiting a combinatorial strategy involving peptide myristoylation and subsequent self-assembly, we successfully converted the host-defense peptide HD5 into efficacious nanobiotics that are fast-acting and have a broad spectrum. HD5 contributes to the innate host defense against various enteropathogens in the gut59-61 and helps maintain intestinal homeostasis by forming a chemical barrier that segregates the gut microbiota from the host epithelium to limit tissue inflammation and microbial translocation.59,

62

Thus, the HD5-myr

nanobiotic may be ideally suited for treating intestinal illnesses caused by enteropathogens. More

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significantly, considering the generality and versatility afforded by our approach, we envision that it could be widely applied to the numerous natural AMP for the creation of nanobiotics. Finally, several excellent properties are combined in this scaffold, including the feasibility of peptide synthesis, the simple one-pot self-assembly procedure for producing the nanobiotics, and the reduced systemic toxicity with a high safety margin in vivo, making the HD5-myr nanobiotic scaffold scalable and promising of future clinical translation.

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Figure Legends Figure 1. (A) Rational engineering of human alpha-defensin 5 (HD5) through peptide myristoylation at the N- or C-terminus and sequential nanoassembly of myristoylated HD5 for generating nanobiotics. (B) Amino acid sequences of HD5, myr-HD5 and HD5-myr with disulfide bonds (brown connective lines) shown. Myristic acid was tethered to HD5 via the amino group of an added N-terminal Gly residue or the ε-amino group of Lys in an extra C-terminal GlyLys linker. The red G represent a myristic acid moiety tethered to N-terminal Gly, and the red K represent a myristic acid moiety tethered to the ε-amino group of Lys. (C) Transmission electron microscopy (TEM) images of parent HD5, and myr-HD5- and HD5-myr-assemblied nanoparticles in DI water. Scale bars, 100 nm. Inserted pictures show the size distribution of myr-HD5- and HD5-myr-assembled nanoparticles determined by dynamic light scattering (DLS).

Figure 2. Antimicrobial activity of HD5 and its myristoylated counterparts, myr-HD5 nanobiotic (NB) and HD5-myr NB. (A-F) Dose-dependent survival of S. aureus, methicillin-resistant S. aureus (MRSA), E. coli, A. baumannii, P. aeruginosa, and K. pneumoniae treated with HD5 and NBs. Bacteria (1×106 CFU/ml) were incubated with the peptides at concentrations varying two-fold from 0.39 µg/ml to 12.5 µg/ml, and the percent bacterial survival (on a logarithmic scale) was calculated as the colonies from treatment wells relative to those from mock-treated control wells. Data from 1.56 to 12.5 µg/ml are shown. (G-H) Survival of E. coli and MRSA (1× 106 CFU/ml) treated for 2 h with 50 µg/ml HD5 and nanobiotics, each at in the presence of 50% fresh mouse serum. Data from three independent experiments are shown as the mean ± SD, and 19


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statistical significance was calculated using one-way ANOVA and Bonferroni’s multiple comparisons test with the following p values: * p

Self-Assembling Myristoylated Human Î±-Defensin 5 as a Next

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