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 β-sheet residues of the native peptide with shorter β-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 β-sheet conformation. These structure–activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins

Rapid Label-free Detection of E. coli using Antimicrobial Peptide Assisted Impedance Spectroscopy

There is an increasing demand for rapid detection of waterborne pathogens to monitor drinking water safety. We demonstrate a compact, label-free sensor array for rapid detection of Escherichia coli (E. coli) in contaminated water samples using antimicrobial peptide assisted impedimetric sensor platform. Interdigitated electrode arrays immobilized with the antimicrobial peptide Colicin V (ColV) were used to screen the affinity towards different bacterial strains by monitoring impedance variations in real-time. This ColV assisted impedance biosensor exhibited high selectivity towards Gram-negative strains particularly towards E. coli strains. This selective detection of E. coli from other strains was observed at 102 cfu mL−1, which is clinically relevant. The sensor can detect E. coli from 102 to 106 cfu mL−1 in water sample at pH 7 to 9. These results show that the antimicrobial peptide ColV assisted impedimetric array is capable of rapid, specific detection of E. coli in contaminated water samples

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

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 alternative 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

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.Science, Faculty ofMicrobiology and Immunology, Department ofReviewedFacultyResearche

Real-time Detection of Breast Cancer Cells Using Peptidefunctionalized 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

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 ml. 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

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-1β. 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.Medicine, Faculty ofMicrobiology and Immunology, Department ofReviewedFacultyPostdoctoralUndergraduat

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 10³ 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

Peptide-Bacteria Interactions using Engineered Surface-Immobilized Peptides from Class IIa Bacteriocins

Specificity of the class IIa bacteriocin Leucocin A (LeuA), an antimicrobial peptide active against Gram-positive bacteria, including Listeria monocytogenes, is known to be dictated by the C-terminal amphipathic helical region, including the extended hairpin-like structure. However, its specificity when attached to a substrate has not been investigated. Exploiting properties of LeuA, we have synthesized two LeuA derivatives, which span the amphipathic helical region of the wild-type LeuA, consisting of 14- (14AA LeuA, CWG­EAF­SAG­VHR­LA) and 24-amino acid residues (24AA LeuA, CSV­NWG­EAF­SAG­VHR­LAN­GGN­GFW). The peptides were purified to >95% purity, as shown by analytical RP-HPLC and mass spectrometry. By including an N-terminal cysteine group, the tailored peptide fragments were readily immobilized at the gold interfaces. The resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicated that the peptides were covalently immobilized in a random helical orientation. The bacterial specificity of the anchored peptide fragments was tested against Gram-positive and Gram-negative bacteria. Our results showed that the adsorbed 14AA LeuA exhibited no specificity toward the bacterial strains, whereas the surface-immobilized 24AA LeuA displayed significant binding toward Gram-positive bacteria with various binding affinities from one strain to another. The 14AA LeuA did not show binding as this fragment is most likely too short in length for recognition by the membrane-bound receptor on the target bacterial cell membrane. These results support the potential use of class IIa bacteriocins as molecular recognition elements in biosensing platforms