Hui-an Yu

Boosting Salt Resistance of Short Antimicrobial Peptides

ABSTRACT The efficacies of many antimicrobial peptides are greatly reduced under high salt concentrations, therefore limiting their use as pharmaceutical agents. Here, we describe a strategy to boost salt resistance and serum stability of short antimicrobial peptides by adding the nonnatural bulky amino acid β-naphthylalanine to their termini. The activities of the short salt-sensitive tryptophan-rich peptide S1 were diminished at high salt concentrations, whereas the activities of its β-naphthylalanine end-tagged variants were less affected.

Easy Strategy To Increase Salt Resistance of Antimicrobial Peptides

ABSTRACT The efficacies of many antimicrobial peptides are greatly reduced under high salt concentrations, limiting their development as pharmaceutical compounds. Here, we describe an easy strategy to increase salt resistance of antimicrobial peptides by replacing tryptophan or histidine residues with the bulky amino acids β-naphthylalanine and β-(4,4′-biphenyl)alanine. The activities of the salt-sensitive peptide P-113 were diminished at high salt concentrations, whereas the activities of its β-naphthylalanine and β-(4,4′-biphenyl)alanine-substituted variant were less affected.

Novel Antimicrobial Peptides with High Anticancer Activity and Selectivity

We describe a strategy to boost anticancer activity and reduce normal cell toxicity of short antimicrobial peptides by adding positive charge amino acids and non-nature bulky amino acid β-naphthylalanine residues to their termini. Among the designed peptides, K4R2-Nal2-S1 displayed better salt resistance and less toxicity to hRBCs and human fibroblast than Nal2-S1 and K6-Nal2-S1. Fluorescence microscopic studies indicated that the FITC-labeled K4R2-Nal2-S1 preferentially binds cancer cells and causes apoptotic cell death. Moreover, a significant inhibition in human lung tumor growth was observed in the xenograft mice treated with K4R2-Nal2-S1. Our strategy provides new opportunities in the development of highly effective and selective antimicrobial and anticancer peptide-based therapeutics.

Efficacy of a novel antimicrobial peptide against periodontal pathogens in both planktonic and polymicrobial biofilm states

UNLABELLED Streptococcus gordonii, Fusobacterium nucleatum and Porphyromonas gingivalis represent the early, middle and late colonizers of the bacterial accretion in dental plaque biofilms. These sessile communities constitute a protected mode of growth that promotes survival in a hostile environment. This study describes a novel and unrecognized role for a synthetic cationic antimicrobial peptide, Nal-P-113, which inhibits and kills periodontal bacteria in planktonic state, inhibits the formation of biofilms and eradicates polymicrobial biofilms. Nal-P-113 is also stable in saliva, serum and saline solution. At a concentration less than 320 μg/mL which is harmless to normal oral cells, Nal-P-113 can kill bacteria in planktonic state. At a concentration of antimicrobial peptide Nal-P-113 (1280 μg/mL) which only causes slight damages to normal oral cells is needed to kill bacteria in biofilm state. It is worth mentioning that this concentration of Nal-P-113 is harmless to rat oral mucosa compared to chlorhexidine. The mechanism of Nal-P-113 inhibiting and killing periodontal bacteria might rely on the abilities to permeabilize and/or to form pores within the cytoplasmic membranes, thus causes the death of bacteria. Here, we provided a novel and stable antimicrobial peptide with very low mammalian cytotoxicity, which can inhibit and kill periodontal bacteria in both planktonic and polymicrobial biofilm states. STATEMENT OF SIGNIFICANCE Nal-P-113 is a potent antimicrobial peptide with strong antimicrobial ability, improved deficiency compared with other antibacterial peptides, and remains stable in phosphate buffered saline, saliva, brain-heart infusion medium and bovine calf serum. Nal-P-113 exhibits a broad spectrum of bacteriocidal activity with excellent eradicating capability on oral pathogens and the respective biofilms. In this study, we used propidium iodide staining, scanning electron microscopy and transmission electron microscopy to confirm that Nal-P-113 can perforate plasmalemma thereby resulting in the death of oral pathogens and disintegrate the respective biofilms. Nal-P-113 also showed effective anti-plaque biofilms and cytotoxicity in the rat periodontitis model. No adverse effects can be observed on the gingivomucosa tissue. In short, the antimicrobial peptide Nal-P-113 presented to be an effective yet have low mammalian cytotoxicity agent with potential application in the clinic. This study provides a proof of concept in applying antimicrobial peptides in the clinical perspective.

A new protocol for high-yield purification of recombinant human CXCL8((3-72))K11R/G31P expressed in Escherichia coli.

The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), binds to both the CXCR1 and CXCR2 receptors with high affinity and the expression levels of CXCL8 are elevated in many inflammatory diseases. Recently, an analogue of human CXCL8, CXCL8((3-72))K11R/G31P (hG31P) has been developed. It has been demonstrated that hG31P is a high affinity antagonist for both CXCR1 and CXCR2. To obtain large quantities of hG31P, we have successfully constructed and expressed hG31P in Escherichia coli. Moreover, we have developed a new protocol for high-yield purification of hG31P and for the removal of lipopolysaccharide (LPS, endotoxin) associated with hG31P due to the expression in E. coli. The purity of hG31P is more than 95% and the final yield is 9.7mg hG31P per gram of cell paste. The purified hG31P was tested by various biological assays. In addition, the structural properties of hG31P were studied by circular dichroism (CD), ultracentrifuge, isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that this purification protocol is very simple and easy to amplify at a large scale. The results of this study will provide an effective route to produce enough hG31P for future clinical studies.

Rational Design of Tryptophan‐Rich Antimicrobial Peptides with Enhanced Antimicrobial Activities and Specificities

Trp‐rich antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp‐rich peptides derived from the C‐terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A2 (PLA2), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK—designated PEM‐2—was found to display improved activity against both Gram‐positive and Gram‐negative bacteria. To improve the antimicrobial activity of PEM‐2 for potential clinical applications further, we determined the solution structure of PEM‐2 bound to membrane‐mimetic dodecylphosphocholine (DPC) micelles by two‐dimensional NMR methods. The DPC micelle‐bound structure of PEM‐2 adopts an α‐helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM‐2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM‐2 could be due to interactions with bacterial membranes. Potent analogues of PEM‐2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies.

Correlations between membrane immersion depth, orientation, and salt-resistance of tryptophan-rich antimicrobial peptides.

The efficacies of many antimicrobial peptides are greatly reduced in the presence of high salt concentrations therefore limiting their development as pharmaceutical compounds. PEM-2-W5K/A9W, a short Trp-rich antimicrobial peptide developed based on the structural studies of PEM-2, has been shown to be highly active against various bacterial strains with less hemolytic activity. Here, correlations between membrane immersion depth, orientation, and salt-resistance of PEM-2 and PEM-2-W5K/A9W have been investigated via solution structure and paramagnetic resonance enhancement studies. The antimicrobial activities of PEM-2-W5K/A9W and PEM-2 against various bacterial and fungal strains including multidrug-resistant and clinical isolates under high salt conditions were tested. The activities of the salt-sensitive peptide PEM-2 were reduced and diminished at high salt concentrations, whereas the activities of PEM-2-W5K/A9W were less affected. The results indicated that the strong salt-resistance of PEM-2-W5K/A9W may arise from the peptide positioning itself deeply into microbial cell membranes and thus able to disrupt the membranes more efficiently.

Role of β-naphthylalanine end-tags in the enhancement of antiendotoxin activities: Solution structure of the antimicrobial peptide S1-Nal-Nal in complex with lipopolysaccharide

Lipopolysaccharide (LPS, endotoxin) is the major component of Gram-negative bacterial outer surface membrane. LPS released from bacteria into bloodstream during infection may cause serious unwanted stimulation of hosts immune system and lead to septic shock of the patient. Recently, we have developed a strategy to increase salt resistance and LPS neutralization of short antimicrobial peptides by adding β-naphthylalanine end-tags to their termini. Herein, correlations between membrane immersion depth, orientation, and antiendotoxin activities of the antimicrobial peptides S1 and S1-Nal-Nal have been investigated via solution structure, paramagnetic resonance enhancement, and saturation transfer difference NMR studies. Unlike the parent peptide S1, S1-Nal-Nal rotated its two terminal β-naphthylalanine residues into the hydrophobic lipid A motif of LPS micelles. The LPS-induced inflammation may then be prohibited by the blocked lipid A motif.

Ultrashort Antimicrobial Peptides with Antiendotoxin Properties

ABSTRACT Release of lipopolysaccharide (LPS) (endotoxin) from bacteria into the bloodstream may cause serious unwanted stimulation of the host immune system. Some but not all antimicrobial peptides can neutralize LPS-stimulated proinflammatory responses. Salt resistance and serum stability of short antimicrobial peptides can be boosted by adding β-naphthylalanine to their termini. Herein, significant antiendotoxin effects were observed in vitro and in vivo with the β-naphthylalanine end-tagged variants of the short antimicrobial peptides S1 and KWWK.

High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli

P-113, which was originally derived from the human saliva protein histatin 5, is a histidine-rich antimicrobial peptide with the sequence AKRHHGYKRKFH. P-113 is currently undergoing phase II clinical trial as a pharmaceutical agent to fight against fungal infections in HIV patients with oral candidiasis. Previously, we developed a new procedure for the high-yield expression and purification of hG31P, an analogue and antagonist of human CXCL8. Moreover, we have successfully removed lipopolysaccharide (LPS, endotoxin) associated with hG31P in the expression with Escherichia coli. In this paper, we have used hG31P as a novel fusion protein for the expression and purification of P-113. The purity of the expressed P-113 is more than 95% and the yield is 4 mg P-113 per liter of E. coli cell culture in Luria-Bertani (LB) medium. The antimicrobial activity of the purified P-113 was tested. Furthermore, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to study the structural properties of P-113. Our results indicate that using hG31P as a fusion protein to obtain large quantities of P-113 is feasible and is easy to scale up for commercial production. An effective way of producing enough P-113 for future clinical studies is evident in this study.