Susanna Su Jan Leong

Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen

Synthetic biology aims to systematically design and construct novel biological systems that address energy, environment, and health issues. Herein, we describe the development of a synthetic genetic system, which comprises quorum sensing, killing, and lysing devices, that enables Escherichia coli to sense and kill a pathogenic Pseudomonas aeruginosa strain through the production and release of pyocin. The sensing, killing, and lysing devices were characterized to elucidate their detection, antimicrobial and pyocin release functionalities, which subsequently aided in the construction of the final system and the verification of its designed behavior. We demonstrated that our engineered E. coli sensed and killed planktonic P. aeruginosa, evidenced by 99% reduction in the viable cells. Moreover, we showed that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%, leading to much sparser and thinner biofilm matrices. These results suggest that E. coli carrying our synthetic genetic system may provide a novel synthetic biology‐driven antimicrobial strategy that could potentially be applied to fighting P. aeruginosa and other infectious pathogens.

Hollow Fiber Membrane Decorated with Ag/MWNTs: Toward Effective Water Disinfection and Biofouling Control

The currently applied disinfection methods during water treatment provide effective solutions to kill pathogens, but also generate harmful byproducts, which are required to be treated with additional efforts. In this work, an alternative and safer water disinfection system consisting of silver nanoparticle/multiwalled carbon nanotubes (Ag/MWNTs) coated on a polyacrylonitrile (PAN) hollow fiber membrane, Ag/MWNTs/PAN, has been developed. Silver nanoparticles of controlled sizes were coated on polyethylene glycol-grafted MWNTs. Ag/MWNTs were then covalently coated on the external surface of a chemically modified PAN hollow fiber membrane to act as a disinfection barrier. A continuous filtration test using E. coli containing feedwater was conducted for the pristine PAN and Ag/MWNTs/PAN composite membranes. The Ag/MWNT coating significantly enhanced the antimicrobial activities and antifouling properties of the membrane against E. coli. Under the continuous filtration mode using E. coli feedwater, the relative flux drop over Ag/MWNTs/PAN was 6%, which was significantly lower than that over the pristine PAN (55%) at 20 h of filtration. The presence of the Ag/MWNT disinfection layer effectively inhibited the growth of bacteria in the filtration module and prevented the formation of biofilm on the surface of the membrane. Such distinctive antimicrobial properties of the composite membrane is attributed to the proper dispersion of silver nanoparticles on the external surface of the membrane, leading to direct contact with bacterium cells.

A photopolymerized antimicrobial hydrogel coating derived from epsilon-poly-L-lysine.

Hydrogels made from epsilon-poly-l-lysine-graft-methacrylamide (EPL-MA) have been found to have impressive wide spectrum antimicrobial activity against both bacteria (specifically Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus) and fungi (specifically Candida albicans and Fusarium solani). The EPL-MA hydrogel also possesses in vitro biocompatibility and EPL-MA solution is relatively non-hemolytic: the concentration needed for onset of human red blood cell (hRBC) hemolysis is 12,500 μg/mL so that the selectivity for the pathogenic microorganisms over hRBCs is 230-1560. Further, EPL-MA hydrogel can be conveniently ultraviolet-immobilized onto plasma-treated plastic surfaces to form thin highly adherent antimicrobial hydrogel coatings for medical devices and implants.

High Potency and Broad-Spectrum Antimicrobial Peptides Synthesized via Ring-Opening Polymerization of α-Aminoacid-N-carboxyanhydrides

Antimicrobial peptides (AMPs), particularly those effective against methicillin-resistant Staphylococcus aureus ( S. aureus ) and antibiotic-resistant Pseudomonas aeruginosa ( P. aeruginosa ), are important alternatives to antibiotics. Typical peptide synthesis methods involving solid-phase sequential synthesis are slow and costly, which are obstacles to their more widespread application. In this paper, we synthesize peptides via ring-opening polymerization of alpha-amino acid N-carboxyanhydrides (NCA) using a transition metal initiator. This method offers high potential for inexpensive synthesis of substantial quantities of AMPs. Lysine (K) was chosen as the hydrophilic amino acid and alanine (A), phenylalanine (F), and leucine (L) as the hydrophobic amino acids. We synthesized five series of AMPs (i.e., P(KA), P(KL), P(KF), P(KAL), and P(KFL)), varied the hydrophobic amino acid content from 0 to 100%, and determined minimal inhibitory concentrations (MICs) against clinically important Gram-negative and Gram-positive bacteria and fungi (i.e., Escherichia coli ( E. coli ), P. aeruginosa , Serratia marcescens ( S. marcescens ), and Candida albicans ( C. albicans ). We found that P(K(10)F(7.5)L(7.5)) and P(K(10)F(15)) show the broadest activity against all five pathogens and have the lowest MICs against these pathogens. For P(K(10)F(7.5)L(7.5)), the MICs against E. coli , P. aeruginosa , S. marcescens , S. aureus , and C. albicans are 31 microg/mL, 31 microg/mL, 250 microg/mL, 31 microg/mL, and 62.5 microg/mL, while for P(K(10)F(15)) the respective MICs are 31 microg/mL, 31 microg/mL, 250 microg/mL, 31 microg/mL, and 125 microg/mL. These are lower than the MICs of many naturally occurring AMPs. The membrane depolarization and SEM assays confirm that the mechanism of microbe killing by P(K(10)F(7.5)L(7.5)) copeptide includes membrane disruption, which is likely to inhibit rapid induction of AMP-resistance in pathogens.

Improvement of biomass properties by pretreatment with ionic liquids for bioconversion process

Cassava pulp residue and rice straw were used as a precursor for pretreatment with ionic liquids to study the effects of pretreatment conditions on product yield and properties. Cassava pulp residue is a potential biomass in the bioconversion process due to it requiring mild pretreatment conditions while providing a high sugar conversion. The maximum sugar conversion and lignin extraction are attained from pretreatment of biomasses with particle size of <38 μm and ionic liquid of 1-Ethyl-3-methylimidazolium acetate at 120°C for 24h. The effectiveness of ionic liquid for biomass pretreatment process follows the sequence: 1-Ethyl-3-methylimidazolium acetate>1-Ethyl-3-methylimidazolium diethyl phosphate>1,3-Dimethylimidazolium methyl sulfate. The increase of pretreatment temperature from 25 to 120°C and decrease of biomass particle size renders higher sugar conversion, lignin extraction and lower crystallinity index. However, pretreatment at temperatures higher than 120°C shows a sharp decline of regenerated biomass yield, sugar conversion and lignin extraction and giving higher crystallinity index at pretreatment temperature of 180°C.

Antimicrobial functionalization of silicone surfaces with engineered short peptides having broad spectrum antimicrobial and salt-resistant properties.

Catheter-associated urinary tract infections (CAUTIs) are often preceded by pathogen colonization on catheter surfaces and are a major health threat facing hospitals worldwide. Antimicrobial peptides (AMPs) are a class of new antibiotics that hold promise in curbing CAUTIs caused by antibiotic-resistant pathogens. This study aims to systematically evaluate the feasibility of immobilizing two newly engineered arginine/lysine/tryptophan-rich AMPs with broad antimicrobial spectra and salt-tolerant properties on silicone surfaces to address CAUTIs. The peptides were successfully immobilized on polydimethylsiloxane and urinary catheter surfaces via an allyl glycidyl ether (AGE) polymer brush interlayer, as confirmed by X-ray photoelectron spectroscopy and water contact angle analyses. The peptide-coated silicone surfaces exhibited excellent microbial killing activity towards bacteria and fungi in urine and in phosphate-buffered saline. Although both the soluble and immobilized peptides demonstrated membrane disruption capabilities, the latter showed a slower rate of kill, presumably due to reduced diffusivity and flexibility resulting from conjugation to the polymer brush. The synergistic effects of the AGE polymer brush and AMPs prevented biofilm formation by repelling cell adhesion. The peptide-coated surface showed no toxicity towards smooth muscle cells. The findings of this study clearly indicate the potential for the development of AMP-based coating platforms to prevent CAUTIs.

Refolding of proteins from inclusion bodies: rational design and recipes.

The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons. Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed.

Development of a catheter functionalized by a polydopamine peptide coating with antimicrobial and antibiofilm properties.

Catheter-associated urinary tract infections (CAUTIs) are the most common hospital-acquired infections worldwide, aggravating the problem of antimicrobial resistance and patient morbidity. There is a need for a potent and robust antimicrobial coating for catheters to prevent these infections. An ideal coating agent should possess high antimicrobial efficacy and be easily and economically conjugated to the catheter surface. In this study, we report a simple yet effective immobilization strategy to tether a potent synthetic antimicrobial peptide, CWR11, onto catheter-relevant surfaces. Polydopamine (PD) was deposited as a thin adherent film onto a polydimethylsiloxane (PDMS) surface to facilitate attachment of CWR11 onto the PD-functionalized polymer. Surface characterization of the CWR11-tethered surfaces confirmed the successful immobilization of peptides onto the PD-coated PDMS. The CWR11-immobilized PDMS slides displayed excellent antimicrobial (significant inhibition of 5×10(4) colony-forming units of CAUTI-relevant microbes) and antibiofilm (∼92% enhanced antibacterial adherence) properties. To assess its clinical relevance, the PD-based immobilization platform was translated onto commercial silicone-coated Foley catheters. The CWR11-impregnated catheter displayed potent bactericidal properties against both Gram-positive and Gram-negative bacteria, and retained its antimicrobial functionality for at least 21days, showing negligible cytotoxicity against human erythrocyte and uroepithelial cells. The outcome of this study demonstrates the proof-of-concept potential of a polydopamine-CWR11-functionalized catheter to combat CAUTIs.

Immobilization Studies of an Engineered Arginine–Tryptophan-Rich Peptide on a Silicone Surface with Antimicrobial and Antibiofilm Activity

With the rapid rise of antibiotic-resistant-device-associated infections, there has been increasing demand for an antimicrobial biomedical surface. Synthetic antimicrobial peptides that have excellent bactericidal potency and negligible cytotoxicity are promising targets for immobilization on these target surfaces. An engineered arginine-tryptophan-rich peptide (CWR11) was developed, which displayed potent antimicrobial activity against a broad spectrum of microbes via membrane disruption, and possessed excellent salt resistance properties. A tethering platform was subsequently developed to tether CWR11 onto a model polymethylsiloxane (PDMS) surface using a simple and robust strategy. Surface characterization assays such as attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), and energy-dispersive X-ray spectroscopy (EDX) confirmed the successful grafting of CWR11 onto the chemically treated PDMS surface. The immobilized peptide concentration was 0.8 ± 0.2 μg/cm(2) as quantitated by sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate (sulfo-SDTB) assay. Antimicrobial assay and cytotoxic investigation confirmed that the peptide-immobilized surface has good bactericidal and antibiofilm properties, and is also noncytotoxic to mammalian cells. Tryptophan-arginine-rich antimicrobial peptides have the potential for antimicrobial protection of biomedical surfaces and may have important clinical applications in patients.

Antimicrobial macromolecules: synthesis methods and future applications

This review article presents important and recent progress in the manufacture and application of antimicrobial macromolecules. Microbial infections continue to endanger human health and pose a great economic burden to society. To resolve this crisis, huge efforts to improve or develop macromolecules that can inhibit pathogens without incurring pathogen resistance are required and actively ongoing. Synthetic antimicrobial macromolecules which include antimicrobial peptides (AMPs), polymers and peptide–polymer hybrids represent a huge class of molecules which can incur effective antimicrobial therapy due to their unique biochemical properties. The use of these antimicrobial macromolecules which target the cytoplasmic membrane of microbes, is a promising approach to lower the propensity of pathogen resistance development. Therefore, huge efforts to synthesize these molecules at scales and purities that enable their structure-function and clinical studies are actively underway. Due to the high cost involved in extracting AMPs from natural sources, biological processes are being developed to economically manufacture AMPs at large scale. Synthetic AMP analogs are also being engineered to further improve antimicrobial potency and lower synthesis cost. Synthetic polymers have also been found to exhibit excellent antimicrobial properties which are comparable to those of natural AMPs. Various antimicrobial polymers have been synthesized based on the amphiphilicity of natural AMPs. Although the facile synthesis of polymers poses no cost problems, numerous synthetic antimicrobial polymers are disadvantaged by high toxicity to mammalian cells due to their non-selectivity. To combine the advantages of AMPs and antimicrobial polymers, peptide-polymer hybrid macromolecules are actively being developed, with a few effective and strongly microbicidal models recently demonstrated. With the advancement of biochemical engineering tools and chemical synthesis methods, these antimicrobial macromolecules can be specifically designed to be highly selective, broad spectrum and biocompatible. In this review, we summarize the recent advances and challenges in the manufacture of these antimicrobial macromolecules. Based on their antimicrobial mechanisms, their applications in addressing challenges associated with infectious disease and antibiotic-resistance are also discussed.