Samuel K. Kutty

Nanoparticle (Star Polymer) Delivery of Nitric Oxide Effectively Negates Pseudomonas aeruginosa Biofilm Formation

Biofilms are increasingly recognized as playing a major role in human infectious diseases, as they can form on both living tissues and abiotic surfaces, with serious implications for applications that rely on prolonged exposure to the body such as implantable biomedical devices or catheters. Therefore, there is an urgent need to develop improved therapeutics to effectively eradicate unwanted biofilms. Recently, the biological signaling molecule nitric oxide (NO) was identified as a key regulator of dispersal events in biofilms. In this paper, we report a new class of core cross-linked star polymers designed to store and release nitric oxide, in a controlled way, for the dispersion of biofilms. First, core cross-linked star polymers were prepared by reversible addition-fragmentation chain transfer polymerization (RAFT) via an arm first approach. Poly(oligoethylene methoxy acrylate) chains were synthesized by RAFT polymerization, and then chain extended in the presence of 2-vinyl-4,4-dimethyl-5-oxazolone monomer (VDM) with N,N-methylenebis(acrylamide) employed as a cross-linker to yield functional core cross-linked star polymers. Spermine was successfully attached to the star core by reaction with VDM. Finally, the secondary amine groups were reacted with NO gas to yield NO-core cross-linked star polymers. The core cross-linked star polymers were found to release NO in a controlled, slow delivery in bacterial cultures showing great efficacy in preventing both cell attachment and biofilm formation in Pseudomonas aeruginosa over time via a nontoxic mechanism, confining bacterial growth to the suspended liquid.

Pyocyanin Facilitates Extracellular DNA Binding to Pseudomonas aeruginosa Influencing Cell Surface Properties and Aggregation

Pyocyanin is an electrochemically active metabolite produced by the human pathogen Pseudomonas aeruginosa. It is a recognized virulence factor and is involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognized as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. eDNA has also been demonstrated to be a major component in establishing P. aeruginosa biofilms. In this study we discovered that production of pyocyanin influences the binding of eDNA to P. aeruginosa PA14 cells, mediated through intercalation of pyocyanin with eDNA. P. aeruginosa cell surface properties including cell size (hydrodynamic diameter), hydrophobicity and attractive surface energies were influenced by eDNA in the presence of pyocyanin, affecting physico-chemical interactions and promoting aggregation. A ΔphzA-G PA14 mutant, deficient in pyocynain production, could not bind with eDNA resulting in a reduction in hydrodynamic diameter, a decrease in hydrophobicity, repulsive physico-chemical interactions and reduction in aggregation in comparison to the wildtype strain. Removal of eDNA by DNase I treatment on the PA14 wildtype strain resulted in significant reduction in aggregation, cell surface hydrophobicity and size and an increase in repulsive physico-chemical interactions, similar to the level of the ΔphzA-G mutant. The cell surface properties of the ΔphzA-G mutant were not affected by DNase I treatment. Based on these findings we propose that pyocyanin intercalation with eDNA promotes cell-to-cell interactions in P. aeruginosa cells by influencing their cell surface properties and physico-chemical interactions.

Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation.

Design, synthesis, and evaluation of fimbrolide-nitric oxide donor hybrids as antimicrobial agents.

Fimbrolides from marine algae have shown promising activity against quorum sensing (QS), a chief regulatory and communication system in bacteria controlling biofilm formation and virulence factor. Nitric oxide (NO) at sublethal concentration has also been reported to induce dispersal of bacterial biofilms and increase their susceptibility toward standard biocides and antibiotics. Therefore, the combination of QS inhibitors and NO donors has the potential to control the development of biofilm and promote their dispersion via a nonbactericidal mechanism. Inspired by these ideas, novel fimbrolide-NO donor hybrid compounds were designed and synthesized. Fimbrolide-NO hybrids 6b, 6f, and 14a were found to be particularly effective as antimicrobials compared to the nonhybrid natural fimbrolides as revealed by bioluminescent P. aeruginosa QS reporter assays and biofilm inhibition assays. Significantly, these fimbrolide-NO hybrids represent the first dual-action antimicrobial agent based on the baterial QS inhibition and NO signaling.

Functional gold nanoparticles for the storage and controlled release of nitric oxide: applications in biofilm dispersal and intracellular delivery†

Gold nanoparticles (size 10 nm) were designed to store and release nitric oxide (NO), by functionalizing their surfaces with functional polymers modified with NO-donor molecules. Firstly, block copolymer chains consisting of poly(oligoethylene glycol methyl ether methacrylate)-b-poly(vinyl benzyl chloride) (P(OEGMA)-b-PVBC)) were prepared using RAFT polymerization. The chloro-functional groups were then reacted with hexylamine, to introduce secondary amine groups to the copolymer chains. The block copolymers were then grafted onto the surface of gold nanoparticles, exploiting the end-group affinity for gold - attaining grafting densities of 0.6 chain per nm2. The secondary amine functional groups were then converted to N-diazeniumdiolate NO donor molecules via exposure to NO gas at high pressure (5 atm). The NO-bearing, gold nanoparticles were characterized using a range of techniques, including transmission electron microscopy, dynamic light scattering (DLS), thermal gravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS). The nanoparticles displayed slow release of the nitric oxide in biological media. Proof of potential utility was then demonstrated in two different application areas: Pseudomonas aeruginosa biofilm dispersal and cancer cell cytotoxicity.

Serratia Secondary Metabolite Prodigiosin Inhibits Pseudomonas aeruginosa Biofilm Development by Producing Reactive Oxygen Species that Damage Biological Molecules

Prodigiosin is a heterocyclic bacterial secondary metabolite belonging to the class of tripyrrole compounds, synthesized by various types of bacteria including Serratia species. Prodigiosin has been the subject of intense research over the last decade for its ability to induce apoptosis in several cancer cell lines. Reports suggest that prodigiosin promotes oxidative damage to double-stranded DNA (dsDNA) in the presence of copper ions and consequently leads to inhibition of cell-cycle progression and cell death. However, prodigiosin has not been previously implicated in biofilm inhibition. In this study, the link between prodigiosin and biofilm inhibition through the production of redox active metabolites is presented. Our study showed that prodigiosin (500 μM) (extracted from Serratia marcescens culture) and a prodigiosin/copper(II) (100 μM each) complex have strong RNA and dsDNA cleaving properties while they have no pronounced effect on protein. Results support a role for oxidative damage to biomolecules by H2O2 and hydroxyl radical generation. Further, it was demonstrated that reactive oxygen species scavengers significantly reduced the DNA and RNA cleaving property of prodigiosin. P. aeruginosa cell surface hydrophobicity and biofilm integrity were significantly altered due to the cleavage of nucleic acids by prodigiosin or the prodigiosin/copper(II) complex. In addition, prodigiosin also facilitated the bactericidal activity. The ability of prodigiosinto cause nucleic acid degradation offers novel opportunities to interfere with extracellular DNA dependent bacterial biofilms.

Synthesis, biological evaluation and structure–activity relationship studies of isoflavene based Mannich bases with potent anti-cancer activity

Phenoxodiol, an analogue of the isoflavone natural product daidzein, is a potent anti-cancer agent that has been investigated for the treatment of hormone dependent cancers. This molecular scaffold was reacted with different primary amines and secondary amines under different Mannich conditions to yield either benzoxazine or aminomethyl substituted analogues. These processes enabled the generation of a diverse range of analogues that were required for structure-activity relationship (SAR) studies. The resulting Mannich bases exhibited prominent anti-proliferative effects against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines. Further cytotoxicity studies against MRC-5 normal lung fibroblast cells showed that the isoflavene analogues were selective towards cancer cells.

N-Acetylglucosamine Inhibits LuxR, LasR and CviR Based Quorum Sensing Regulated Gene Expression Levels

N-acetyl glucosamine, the monomer of chitin, is an abundant source of carbon and nitrogen in nature as it is the main component and breakdown product of many structural polymers. Some bacteria use N-acyl-L-homoserine lactone (AHL) mediated quorum sensing (QS) to regulate chitinase production in order to catalyze the cleavage of chitin polymers into water soluble N-acetyl-D-glucosamine (NAG) monomers. In this study, the impact of NAG on QS activities of LuxR, LasR, and CviR regulated gene expression was investigated by examining the effect of NAG on QS regulated green fluorescent protein (GFP), violacein and extracellular chitinase expression. It was discovered that NAG inhibits AHL dependent gene transcription in AHL reporter strains within the range of 50–80% reduction at low millimolar concentrations (0.25–5 mM). Evidence is presented supporting a role for both competitive inhibition at the AHL binding site of LuxR type transcriptional regulators and catabolite repression. Further, this study shows that NAG down-regulates CviR induced violacein production while simultaneously up-regulating CviR dependent extracellular enzymes, suggesting that an unknown NAG dependent regulatory component influences phenotype expression. The quorum sensing inhibiting activity of NAG also adds to the list of compounds with known quorum sensing inhibiting activities.

Development of Fimbrolides, Halogenated Furanones and their Derivatives as Antimicrobial Agents

Traditional treatment for bacterial infection is based on compounds that target bacterial viability or growth. However, a major concern with this approach is the frequent development of drug-resistant mutants. The discovery of bacterial quorum sensing (QS) systems, which control fundamental processes involved in bacterial physiology and virulence, has opened new avenues for the development of antimicrobial agents for the control of bacterial infections. Fimbrolides isolated from Australian native marine alga Delisea pulchra are excellent examples of QS inhibitors provided by nature. Fimbrolides and their analogues exhibit excellent QS inhibitory activity without interfering with bacterial growth, and thus offer promising targets for development of new strategies to control microbial colonisation of surfaces. This chapter describes the types of natural fimbrolides, their biosynthesis, and synthesis of related halogenated furanone and dihydropyrrolone analogues, as well as their biological activities and applications as antimicrobial coatings for the prevention of bacterial infections.

Nitric Oxide Donors as Antimicrobial Agents

Abstract Nitric oxide (NO) is a simple yet vital biological molecule. It plays a significant role in a wide array of biological processes crucial for proper maintenance of human body, such as host defense, cardiovascular regulation, smooth muscle relaxation, platelet aggregation, neurotransmission, and wound healing. In addition to the diverse physiological functions, NO is also a key bacterial signaling molecule and regulates diverse downstream processes in bacteria such as biofilm dispersal, motility, and virulence. NO can thus be used to regulate different pathways in bacteria and thereby control pathogenicity and biofilm formation of bacteria. This chapter will discuss the current understanding of the role of NO in bacterial life cycle, the potential use of NO to control bacterial infection, pathogenicity and biofilm, as well as the development of NO donors and NO hybrids as antimicrobial agents for future treatment of bacterial infections.