Impacts of PEGylation and Glycosylation on the Biological Properties of Host Defense Peptide IDR1018.

The multifunctional properties of host defense peptides (HDPs) make them promising drug candidates to tackle bacterial infections and tissue inflammation. However, these peptides tend to aggregate and can harm host cells at high doses, potentially limiting their clinical use and applications. In this study, we explored the influences of both pegylation and glycosylation on the biocompatibility and biological properties of HDPs, particularly the innate defense regulator IDR1018. Two peptide conjugates were designed by attaching either polyethylene glycol (PEG6) or a glucose moiety to the peptide towards the N-terminus. Significantly, both derivatives reduced the aggregation, hemolysis, and cytotoxicity of the parent peptide by orders of magnitude. In addition, while the pegylated conjugate, PEG6-IDR1018, retained an excellent immunomodulatory profile, similar to that observed for IDR1018 itself, the glycosylated conjugate, Glc-IDR1018, significantly outperformed the parent peptide in inducing anti-inflammatory mediators, MCP1 and IL-1RA and in suppressing the level of lipopolysaccharide-induced proinflammatory cytokine IL-1ß. Conversely, the conjugates led to a partial reduction in antimicrobial and antibiofilm activity. These findings underline the impacts of both pegylation and glycosylation on the biological properties of the HDP IDR1018 and indicate the potential of glycosylation to enhance the design of highly effective immunomodulatory peptides.

Antibiofilm and immunomodulatory resorbable nanofibrous filing for dental pulp regenerative procedures.

Multifunctional scaffolds with host defense peptides designed for regenerative endodontics are desirable nanobiotechnological tools for dentistry. Here, different scaffolds were tested for use during the pulp revascularization process, including poly(vinyl alcohol)-PVA hydrogels or resins, collagen hydrogels and poly(vinyl alcohol) PVA/Chitosan (PVA/CS) nanofibers. Based on time to degradation (21 days), nanofibers were chosen to be incorporated with ciprofloxacin and IDR-1002 (each at 50 mg/g). Nanofibers containing ciprofloxacin and IDR-1002 had anti-biofilm activity against Enterococcus faecalis, Staphylococcus aureus and a multispecies oral biofilm, besides anti-inflammatory activities. The in vivo subcutaneous tissue response to tooth fragments filled with nanofibers demonstrated a pulp-like tissue formation, when compared to empty teeth fragments. Thus, we designed a strong antimicrobial, immunomodulatory and regenerative candidate for pulp revascularization and regeneration procedures.

Host Defense Peptide-Mimicking Polymers and Polymeric-Brush-Tethered Host Defense Peptides: Recent Developments, Limitations, and Potential Success.

Amphiphilic antimicrobial polymers have attracted considerable interest as structural mimics of host defense peptides (HDPs) that provide a broad spectrum of activity and do not induce bacterial-drug resistance. Likewise, surface engineered polymeric-brush-tethered HDP is considered a promising coating strategy that prevents infections and endows implantable materials and medical devices with antifouling and antibacterial properties. While each strategy takes a different approach, both aim to circumvent limitations of HDPs, enhance physicochemical properties, therapeutic performance, and enable solutions to unmet therapeutic needs. In this review, we discuss the recent advances in each approach, spotlight the fundamental principles, describe current developments with examples, discuss benefits and limitations, and highlight potential success. The review intends to summarize our knowledge in this research area and stimulate further work on antimicrobial polymers and functionalized polymeric biomaterials as strategies to fight infectious diseases.

Multifunctional Antibiotic-Host Defense Peptide Conjugate Kills Bacteria, Eradicates Biofilms, and Modulates the Innate Immune Response.

Effective anti-infective therapies are required to offset the rise in antibiotic resistance. A novel vancomycin-innate defense regulator conjugate (V-IDR1018) was constructed with multimodal functionality, including bacterial killing, biofilm eradication, and immune modulation. The conjugate killed bacteria within 30 min, exhibited potent activity against persister cells, and showed no susceptibility to antimicrobial resistance in tissue culture assays. Additionally, it stimulated the release of chemokine MCP-1 and anti-inflammatory cytokine IL-10 and suppressed pro-inflammatory IL-1ß from lipopolysaccharide-stimulated white blood cells. The conjugate demonstrated ∼90% eradication efficacy when assessed against the MRSA biofilm formed on an organoid human skin equivalent. Similarly, when evaluated in a murine, high-density skin abscess infection model using MRSA or Staphylococcus epidermidis, the conjugate decreased dermonecrosis and reduced bacterial load. The exceptional in vitro and in vivo efficacy of the conjugate, in addition to its safety profile, makes it a valuable candidate to treat complex infectious diseases.

Assessing biofilm inhibition and immunomodulatory activity of small amounts of synthetic host defense peptides synthesized using SPOT-array technology.

Peptides are promising drug candidates because of their diversity, biocompatibility and spectrum of activities. Here, we describe a protocol for high-throughput screening of SPOT-peptide arrays to assess the antibiofilm, antimicrobial and immunomodulatory activities of synthetic peptides. It is a Protocol Extension of our previous Nature Protocols article, which describes the synthesis of SPOT-peptide arrays and assays for screening antimicrobial activity. This latest protocol allows the simultaneous assessment of hundreds of synthetic host defense peptides to define their overall activity profiles and identify candidate sequences that are suitable for further characterization and development as anti-infectives. When coupled with the SPOT-array technology for peptide synthesis, the described procedures are rapid, inexpensive and straightforward for peptide library screening. The protocols can be implemented in most microbiology or immunology research laboratories without the need for specialists. The time to complete each step ranges between 1 and 4 h with overnight pauses, and datasets related to the antibiofilm and immunomodulatory activities of a large set of peptide sequences can be generated in a few days.

Host Defense Peptide-Mimicking Amphiphilic ß-Peptide Polymer (Bu:DM) Exhibiting Anti-Biofilm, Immunomodulatory, and in Vivo Anti-Infective Activity.

Therapeutic options to treat multidrug resistant bacteria, especially when present in biofilms, are limited due to their high levels of antibiotic resistance. Here, we report the anti-biofilm and immunomodulatory activities of the host defense peptide (HDP)-mimicking ß-peptide polymer (20:80 Bu:DM) and investigated its activity in vivo. The polymer outperformed antibiotics in the removal and reduction of the viability of established biofilms, achieving a maximum activity of around 80% reduction in viability. Interestingly the polymer also exhibited HDP-like immunomodulation in inducing chemokines and anti-inflammatory cytokines and suppressing lipopolysaccharide-induced proinflammatory cytokines. When tested in a murine, high-density skin infection model using P. aeruginosa LESB58, the polymer was effective in diminishing abscess size and reducing bacterial load. This study demonstrates the dual functionality of HDP-mimicking ß-peptide polymers in inhibiting biofilms and modulating innate immunity, as well as reducing tissue dermonecrosis.

Cyclic Derivative of Host-Defense Peptide IDR-1018 Improves Proteolytic Stability, Suppresses Inflammation, and Enhances In Vivo Activity.

Host-defense peptides have drawn significant attention as new drugs or drug adjuvants to combat multidrug-resistant bacteria. In this study, we report the development of cyclic derivatives of the immunomodulatory and antibiofilm innate defense regulator peptide (IDR)-1018 based on three different synthetic strategies including head-to-tail cyclization (C1), side-chain-to-tail cyclization (C2), and a disulfide bond cross-linkage (C3). The generated mimetics showed enhanced proteolytic stability and reduced aggregation in vitro and in vivo. The C2 derivative exhibited exceptional ability to suppress inflammation and significantly reduce bacterial loads in a high-density Staphylococcus aureus murine skin infection model. The findings describe different routes to the creation of enzymatically stable mimetics of IDR-1018 and identify a promising new cyclic analogue against bacterial infections.

Modified cantilever arrays improve sensitivity and reproducibility of nanomechanical sensing in living cells.

Mechanical signaling involved in molecular interactions lies at the heart of materials science and biological systems, but the mechanisms involved are poorly understood. Here we use nanomechanical sensors and intact human cells to provide unique insights into the signaling pathways of connectivity networks, which deliver the ability to probe cells to produce biologically relevant, quantifiable and reproducible signals. We quantify the mechanical signals from malignant cancer cells, with 10 cells per ml in 1000-fold excess of non-neoplastic human epithelial cells. Moreover, we demonstrate that a direct link between cells and molecules creates a continuous connectivity which acts like a percolating network to propagate mechanical forces over both short and long length-scales. The findings provide mechanistic insights into how cancer cells interact with one another and with their microenvironments, enabling them to invade the surrounding tissues. Further, with this system it is possible to understand how cancer clusters are able to co-ordinate their migration through narrow blood capillaries.

Portable Nanofiber-Light Addressable Potentiometric Sensor for Rapid Escherichia coli Detection in Orange Juice.

The growing need to prevent pathogen outbreaks is irrefutable in the case of the food industry. Early detection in products, especially beverages, contaminated with bacterial strains is vital to avoid infected foods from reaching the consumer. If E. coli is pesent in such foods, it can cause infections. It can also be an indicator of the existence of other harmful coliforms. In this study, we have investigated the detection of Escherichia coli ( E. coli) in orange juice using a portable nanofiber-light addressable potentiometric sensor (NF-LAPS). We have chosen electrospun pH-sensitive poly(vinyl alcohol)/poly(acrylic acid) (PVA/PAA) hydrogel NFs as the sensitive layer. The successful detection of E. coli was reported with the NF-LAPS in less than 1 h. The limit of detection (LOD) measured in the sensor is found to be102 CFU/mL. We have confirmed the selectivity of the biosensor toward E. coli by examining the response of the NF-LAPS against Salmonella typhimurium ( S. typhi), also commonly found in orange juice. Despite the complex nature of orange juice, the response of the biosensor is in no way affected while orange juice is tested as is.

Metabolic Study of Cancer Cells Using a pH Sensitive Hydrogel Nanofiber Light Addressable Potentiometric Sensor.

We report a simple, fast, and cost-effective approach that measures cancer cell metabolism and their response to anticancer drugs in real time. Using a Light Addressable Potentiometric Sensor integrated with pH sensitive hydrogel nanofibers (NF-LAPS), we detect localized changes in pH of the media as cancer cells consume glucose and release lactate. NF-LAPS shows a sensitivity response of 74 mV/pH for cancer cells. Cancer cells (MDA MB231) showed a response of ∼0.4 unit change in pH compared to virtually no change observed for normal cells (MCF10A). We also observed a drop in pH for the multidrug-resistant cancer cells (MDA-MB-435MDR) in the presence of doxorubicin. However, inhibition of the metabolic enzymes such as hexokinase and lactate dehydrogenase-A suggested an improvement in the efficacy of doxorubicin by decreasing the level of acidification. This approach, based on extracellular acidification, enhances our understanding of cancer cell metabolic modes and their response to chemotherapies, which will help in the development of better treatments, including choice of drugs and dosages.

Proteolytically Stable Cyclic Decapeptide for Breast Cancer Cell Targeting.

Starting with a previously reported linear breast cancer targeting decapeptide WxEAAYQkFL, here we report the synthesis of a novel cyclic peptide analogue cyclic WXEAAYQkFL. The N- to C-terminus amide cyclized peptide with one d-amino acid (k) displayed higher uptake by breast cancer cells, with minimal uptake by the noncancerous cells compared to the linear peptide with two d-amino acids (x and k), and was stable toward proteolytic degradation. When immobilized on gold microcantilever surface, the cyclic peptide was able to capture breast cancer cells specifically and sense samples with ≥25 cancer cells/mL. Animal studies using mice carrying orthotopic breast MDA-MB-231 tumors showed that the cyclic peptide preferentially accumulates in tumor (2 h after injection) and is rapidly cleared from all other organs except kidneys and liver. The study highlights the discovery of a novel proteolytically stable cyclic peptide that can be used for targeted drug delivery or for enumerating circulating breast tumor cells.

Bacterial Detection Using Peptide-Based Platform and Impedance Spectroscopy.

Antimicrobial peptides have the ability to function as bio-recognition elements in the detection of bacteria. For instance, we showed that Leucocin A, an antimicrobial peptide from class IIa bacteriocins, binds gram-positive Listeria monocytogenes with higher affinity than other gram-positive bacteria like S. aureus, L. innocua, and E. faecalis. The binding was detected using impedance spectroscopy when Leucocin A immobilized on impedance electrodes binds bacteria from a sample. Here we highlight the strength of utilizing Leucocin A as a bio-recognition probe in biosensor platforms and provide details on its application in real-time bacterial detection using electrochemical impedance spectroscopy. A simple new generation impedance array analyzer is utilized that works at very low frequencies to identify interactions between peptide and the target bacteria.

Breast Cancer Targeting Peptide Binds Keratin 1: A New Molecular Marker for Targeted Drug Delivery to Breast Cancer.

The biomarkers or receptors expressed on cancer cells and the targeting ligands with high binding affinity for biomarkers play a key role in early detection and treatment of breast cancer. The breast cancer targeting peptide p160 (12-mer) and its enzymatically stable analogue 18-4 (10-mer) showed marked potential for breast cancer drug delivery using cell studies and animal models. Herein, we used affinity purification, liquid chromatography-tandem mass spectrometry, and proteomics to identify keratin 1 (KRT1) as the target receptor highly expressed on breast cancer cells for p160 peptide(s). Western blot and immunocytochemistry in MCF-7 breast cancer cells confirmed the identity of KRT1. We demonstrate that the p160 or 18-4 binding to MCF-7 breast cancer cells is dependent on the expression of KRT1, and we confirm peptide-KRT1 binding specificity using SPR experiments (Kd ∼ 1.1 µM and 0.98 µM for p160 and 18-4, respectively). Furthermore, we assessed the ability of peptide 18-4 to improve the cellular uptake and anticancer activity of a pro-apoptotic antimicrobial peptide, microcin J25 (MccJ25), in breast cancer cells. A covalent conjugate of peptide 18-4 with MccJ25 showed preferential cytotoxicity toward breast cancer cells with minimal cytotoxicity against normal HUVEC cells. The conjugate inhibited the growth of MDA-MB-435 MDR multidrug-resistant cells with an IC50 comparable to that of nonresistant cells. Conjugation improved selective cellular uptake of MccJ25, and the conjugate triggered cancer cell death by apoptosis. Our findings establish KRT1 as a new marker for breast cancer targeting. Additionally, it pinpoints the potential use of antimicrobial lasso peptides as a novel class of anticancer therapeutics.

Microfluidic cantilever detects bacteria and measures their susceptibility to antibiotics in small confined volumes.

In the fight against drug-resistant bacteria, accurate and high-throughput detection is essential. Here, a bimaterial microcantilever with an embedded microfluidic channel with internal surfaces chemically or physically functionalized with receptors selectively captures the bacteria passing through the channel. Bacterial adsorption inside the cantilever results in changes in the resonance frequency (mass) and cantilever deflection (adsorption stress). The excitation of trapped bacteria using infrared radiation (IR) causes the cantilever to deflect in proportion to the infrared absorption of the bacteria, providing a nanomechanical infrared spectrum for selective identification. We demonstrate the in situ detection and discrimination of Listeria monocytogenes at a concentration of single cell per µl. Trapped Escherichia coli in the microchannel shows a distinct nanomechanical response when exposed to antibiotics. This approach, which combines enrichment with three different modes of detection, can serve as a platform for the development of a portable, high-throughput device for use in the real-time detection of bacteria and their response to antibiotics.

Real-time Detection of Breast Cancer Cells Using Peptide-functionalized Microcantilever Arrays.

Ligand-directed targeting and capturing of cancer cells is a new approach for detecting circulating tumor cells (CTCs). Ligands such as antibodies have been successfully used for capturing cancer cells and an antibody based system (CellSearch(®)) is currently used clinically to enumerate CTCs. Here we report the use of a peptide moiety in conjunction with a microcantilever array system to selectively detect CTCs resulting from cancer, specifically breast cancer. A sensing microcantilever, functionalized with a breast cancer specific peptide 18-4 (WxEAAYQrFL), showed significant deflection on cancer cell (MCF7 and MDA-MB-231) binding compared to when exposed to noncancerous (MCF10A and HUVEC) cells. The peptide-functionalized microcantilever allowed efficient capture and detection of cancer cells in MCF7 spiked human blood samples emulating CTCs in human blood. A detection limit of 50-100 cancer cells mL(-1) from blood samples was achieved with a capture yield of 80% from spiked whole blood samples. The results emphasize the potential of peptide 18-4 as a novel peptide for capturing and detecting cancer cells in conjunction with nanomechanical cantilever platform. The reported peptide-based cantilever platform represents a new analytical approach that can lead to an alternative to the various detection platforms and can be leveraged to further study CTCs.

Peptide Bacteriocins--Structure Activity Relationships.

With the growing concerns in the scientific and health communities over increasing levels of antibiotic resistance, antimicrobial peptide bacteriocins have emerged as promising alternatives to conventional small molecule antibiotics. A substantial attention has recently focused on the utilization of bacteriocins in food preservation and health safety. Despite the fact that a large number of bacteriocins have been reported, only a few have been fully characterized and structurally elucidated. Since knowledge of the molecular structure is a key for understanding the mechanism of action and therapeutic effects of peptide, we centered our focus in this review on the structure-activity relationships of bacteriocins with a particular focus in seven bacteriocins, namely, nisin, microcin J25, microcin B17, microcin C, leucocin A, sakacin P, and pediocin PA-1. Significant structural changes responsible for the altered activity of the recent bacteriocin analogues are discussed here.

Impedimetric detection of pathogenic Gram-positive bacteria using an antimicrobial peptide from class IIa bacteriocins.

Real-time, label-free detection of Gram-positive bacteria with high selectivity and sensitivity is demonstrated using an interdigitated impedimetric array functionalized with naturally produced antimicrobial peptide from class IIa bacteriocins. The antimicrobial peptide, leucocin A, was chemically synthesized and covalently immobilized on interdigitated gold microelectrodes via the interaction between the C-terminal carboxylic acid of the peptide and free amines of a preattached thiolated linker. Exposing the peptide sensor to various concentrations of Gram-positive bacteria generated reproducible impedance spectra that detected peptide-bacteria interactions at a concentration of 1 cell/µL. The peptide sensor also selectively detected Listeria monocytogenes from other Gram-positive strains at a concentration of 10(3) cfu mL(-1). The study highlights that short peptide ligands from bacteriocin class offer high selectivity in bacterial detection and can be used in developing a robust, portable biosensor device to efficiently detect pathogenic Gram-positive bacteria in food samples.

Surface-conjugated antimicrobial peptide leucocin a displays high binding to pathogenic gram-positive bacteria.

Leucocin A, a representative class IIa bacteriocin, is a ribosomally synthesized antimicrobial peptide (AMP) that displays potent activity against specific gram-positive bacteria. The antibacterial activity of such peptides is preceded by the binding event that can be utilized for studying specific peptide-bacteria interactions. In this study, 37-residue Leucocin A (LeuA) was synthesized using solid-phase peptide synthesis and covalently immobilized on gold substrates from either the N- or C-terminal. Both the peptide monolayers on gold substrates were incubated separately with five strains of gram-positive bacteria and displayed differential binding to different strains with highest binding to pathogenic Listeria monocytogenes . The C-terminally immobilized LeuA showed higher bacterial binding compared to the N-terminally attached LeuA. The full length immobilized LeuA (37-residue) was active as well as displayed higher bacterial binding (73 ± 6 bacteria/100 µm(2)) compared to 24-residue inactive LeuA fragment (40 ± 8 bacteria/100 µm(2)) from the C-terminal region. The high and specific bacterial binding ability of LeuA functionalized surfaces support the potential use of class IIa bacteriocins in antimicrobial peptide-based diagnostic platforms.

Design, synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues.

Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure-activity relationship in the N-terminal domain of these peptides, three analogues (1-3) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal ß-sheet residues of the native peptide with shorter ß-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into ß-sheet conformation. These structure-activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.