Facile fabrication of antibacterial and antiviral perhydrolase-polydopamine composite coatings.

In situ generation of antibacterial and antiviral agents by harnessing the catalytic activity of enzymes on surfaces provides an effective eco-friendly approach for disinfection. The perhydrolase (AcT) from Mycobacterium smegmatis catalyzes the perhydrolysis of acetate esters to generate the potent disinfectant, peracetic acid (PAA). In the presence of AcT and its two substrates, propylene glycol diacetate and H2O2, sufficient and continuous PAA is generated over an extended time to kill a wide range of bacteria with the enzyme dissolved in aqueous buffer. For extended self-disinfection, however, active and stable AcT bound onto or incorporated into a surface coating is necessary. In the current study, an active, stable and reusable AcT-based coating was developed by incorporating AcT into a polydopamine (PDA) matrix in a single step, thereby forming a biocatalytic composite onto a variety of surfaces. The resulting AcT-PDA composite coatings on glass, metal and epoxy surfaces yielded up to 7-log reduction of Gram-positive and Gram-negative bacteria when in contact with the biocatalytic coating. This composite coating also possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes. The single-step approach enables rapid and facile fabrication of enzyme-based disinfectant composite coatings with high activity and stability, which enables reuse following surface washing. As a result, this enzyme-polymer composite technique may serve as a general strategy for preparing antibacterial and antiviral surfaces for applications in health care and common infrastructure safety, such as in schools, the workplace, transportation, etc.

Antimicrobial effects of positively charged, conductive electrospun polymer fibers.

In recent years, electrospun polymer fibers have gained attention for various antibacterial applications. In this work, the effect of positively charged polymer fiber mats as antibacterial gauze is studied using electrospun poly(caprolactone) and polyaniline nanofibers. Chloroxylenol, an established anti-microbial agent is used for the first time as a secondary dopant to polyaniline during the electrospinning process to make the surface of the polyaniline fiber positively charged. Both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli are used to investigate the antibacterial activity of the positively charged and uncharged polymer surfaces. The results surprisingly show that the polyaniline surface can inhibit the growth of both bacteria even when chloroxylenol is used below its minimum inhibitory concentration. This study provides new insights allowing the better understanding of dopant-based, intrinsically conducting polymer surfaces for use as antibacterial fiber mats.

Designer DNA architecture offers precise and multivalent spatial pattern-recognition for viral sensing and inhibition.

DNA, when folded into nanostructures with a specific shape, is capable of spacing and arranging binding sites into a complex geometric pattern with nanometre precision. Here we demonstrate a designer DNA nanostructure that can act as a template to display multiple binding motifs with precise spatial pattern-recognition properties, and that this approach can confer exceptional sensing and potent viral inhibitory capabilities. A star-shaped DNA architecture, carrying five molecular beacon-like motifs, was constructed to display ten dengue envelope protein domain III (ED3)-targeting aptamers into a two-dimensional pattern precisely matching the spatial arrangement of ED3 clusters on the dengue (DENV) viral surface. The resulting multivalent interactions provide high DENV-binding avidity. We show that this structure is a potent viral inhibitor and that it can act as a sensor by including a fluorescent output to report binding. Our molecular-platform design strategy could be adapted to detect and combat other disease-causing pathogens by generating the requisite ligand patterns on customized DNA nanoarchitectures.

Modular Assembly of Unique Chimeric Lytic Enzymes on a Protein Scaffold Possessing Anti-Staphylococcal Activity.

Lytic enzymes have been considered as potential alternatives to antibiotics. These enzymes, particularly those that target Gram-positive bacteria, consist of modular cell wall-binding and catalytic domains, which can be shuffled with those of other lytic enzymes to produce unnatural chimeric enzymes. In this work, we report the in vitro shuffling of two different modular domains using a protein self-assembly methodology. Catalytic domains (CD) and cell wall-binding domains (BD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1), respectively, were genetically site-specifically biotinylated and assembled with streptavidin to generate 23 permuted chimeras. The specific assembly of a CD (3 equiv) and a BD (1 equiv) from Lst and SA1, respectively [CDL-BDS (3:1)], on a streptavidin scaffold yielded high lytic activity against S. aureus (at least 5.6 log reduction), which was higher than that obtained with either native Lst or SA1 alone. Moreover, at 37 °C, the initial rate of cell lysis was over 3-fold higher than that with free Lst, thereby revealing the unique catalytic properties of the chimeric proteins. In vitro self-assembly of functional domains from modular lytic enzymes on a protein scaffold likely expands the repertoire of bactericidal enzymes with improved activities.

Sensitive multiplex detection of whole bacteria using self-assembled cell binding domain complexes.

Detecting bacterial cells at low levels is critical in public health, the food industry and first response. Current processes typically involve laborious cell lysis and genomic DNA extraction to achieve 100-1000 CFU mL-1 levels for detecting gram-positive bacteria. As an alternative to DNA-based methods, cell wall binding domains (CBDs) derived from lysins having a modular structure with an N-terminal catalytic domain and a C-terminal CBD, can be used to detect bacterial pathogens as a result of their exceptionally specific binding to target bacteria with great avidity. We have developed a highly sensitive method for multiplex detection of whole bacterial cells using self-assembled CBD complexes. Self-assembled CBD-SA-reporter complexes were generated using streptavidin (SA), biotin-CBDs, and biotinylated reporters, such as glucose oxidase (GOx) and specific DNA sequences. The simultaneous detection of three test bacteria, Staphylococcus aureus, Bacillus anthracis-Sterne, and Listeria innocua cells in PBS could be accomplished with a 96-well plate-based sandwich method using CBD-SA-GOx complex-coupled spectrophotometric assay to achieve a detection limit of >100 CFU mL-1. To achieve greater detection sensitivity, we used CBD-SA-DNA complexes and qPCR of specific DNA barcodes selectively bound to the surface of target bacterial cells, which resulted in a detection sensitivity as low as 1-10 CFU mL-1 without cross-reactivity. This sensitive multiplex detection of bacterial pathogens using both CBD-SA-GOx and CBD-SA-DNA complexes has the potential to be quickly combined with point-of-care compatible diagnostics for the rapid detection of pathogens in test samples.

Unprotonated Short-Chain Alkylamines Inhibit Staphylolytic Activity of Lysostaphin in a Wall Teichoic Acid-Dependent Manner.

Lysostaphin (Lst) is a potent bacteriolytic enzyme that kills Staphylococcus aureus, a common bacterial pathogen of humans and animals. With high activity against both planktonic cells and biofilms, Lst has the potential to be used in industrial products, such as commercial cleansers, for decontamination. However, Lst is inhibited in the presence of monoethanolamine (MEA), a chemical widely used in cleaning solutions and pharmaceuticals, and the underlying mechanism of inhibition remains unknown. In this study, we examined the cell binding and killing capabilities of Lst against S. aureus ATCC 6538 in buffered salt solution with MEA at different pH values (7.5 to 10.5) and discovered that only the unprotonated form of MEA inhibited Lst binding to the cell surface, leading to low Lst activity, despite retention of its secondary structure. This reduced enzyme activity could be largely recovered via a reduction in wall teichoic acid (WTA) biosynthesis through tunicamycin treatment, indicating that the suppression of Lst activity was dependent on the presence and amount of WTA. We propose that the decreased cell binding and killing capabilities of Lst are associated with the influence of uncharged MEA on the conformation of WTA. A similar effect was confirmed with other short-chain alkylamines. This study offers new insight into the impact of short-chain alkylamines on both Lst and WTA structure and function and provides guidance for the application of Lst in harsh environments.IMPORTANCE Lysostaphin (Lst) effectively and selectively kills Staphylococcus aureus, the bacterial culprit of many hospital- and community-acquired skin and respiratory infections and food poisoning. Lst has been investigated in animal models and clinical trials, industrial formulations, and environmental settings. Here, we studied the mechanistic basis of the inhibitory effect of alkylamines, such as monoethanolamine (MEA), a widely used chemical in commercial detergents, on Lst activity, for the potential incorporation of Lst in disinfectant solutions. We have found that protonated MEA has little influence on Lst activity, while unprotonated MEA prevents Lst from binding to S. aureus cells and hence dramatically decreases the enzyme's bacteriolytic efficacy. Following partial removal of the wall teichoic acid, an important component of the bacterial cell envelope, the inhibitory effect of unprotonated MEA on Lst is reduced. This phenomenon can be extended to other short-chain alkylamines. This mechanistic report of the impact of alkylamines on Lst functionality will help guide future applications of Lst in disinfection and decontamination of health-related commercial products.

Selective Killing of Pathogenic Bacteria by Antimicrobial Silver Nanoparticle-Cell Wall Binding Domain Conjugates.

Broad-spectrum antibiotics indiscriminately kill bacteria, removing nonpathogenic microorganisms and leading to evolution of antibiotic resistant strains. Specific antimicrobials that could selectively kill pathogenic bacteria without targeting other bacteria in the natural microbial community or microbiome may be able to address this concern. In this work, we demonstrate that silver nanoparticles, suitably conjugated to a selective cell wall binding domain (CBD), can efficiently target and selectively kill bacteria. As a relevant example, CBDBA from Bacillus anthracis selectively bound to B. anthracis in a mixture with Bacillus subtilis, as well in a mixture with Staphylococcus aureus. This new biologically-assisted hybrid strategy, therefore, has the potential to provide selective decontamination of pathogenic bacteria with minimal impact on normal microflora.

In vitro gene expression-coupled bacterial cell chip for screening species-specific antimicrobial enzymes.

Targeting infectious bacterial pathogens is important for reducing the evolution of antibiotic-resistant bacteria and preserving the endogenous human microbiome. Cell lytic enzymes including bacteriophage endolysins, bacterial autolysins, and other bacteriolysins are useful antibiotic alternatives due to their exceptional target selectivity, which may be used to lysins rapidly kill target bacteria and their high specificity permit the normal commensal microflora to be left undisturbed. Genetic information of numerous lysins is currently available, but the identification of their antimicrobial function and specificity has been limited because most lysins are often poorly expressed and exhibit low solubilities. Here, we report the development of bacterial cell chip for rapidly accessing the function of diverse genes that are suggestive of encoding lysins. This approach can be used to evaluate rapidly the species-specific antimicrobial activity of diverse lysins synthesized from in vitro transcription and translation (TNT) of plasmid DNA. In addition, new potent lysins can be assessed that are not expressed in hosts and display low solubility. As a result of evaluating the species-specific antimicrobial function of 11 (un)known lysins with an in vitro TNT-coupled bacterial cell chip, a potent recombinant lysin against Staphylococcus strains, SA1, was identified. The SA1 was highly potent against not only S. aureus, but also both lysostaphin-resistant S. simulans and S. epidermidis cells. To this end, the SA1 may be applicable to treat both methicillin-resistant S. aureus (MRSA) and lysostaphin-resistant MRSA mutants. Biotechnol. Bioeng. 2017;114: 1648-1657. © 2017 Wiley Periodicals, Inc.

Enhancement of cellulolytic enzyme activity by clustering cellulose binding domains on nanoscaffolds.

Cellulose, one of the most abundant carbon resources, is degraded by cellulolytic enzymes called cellulases. Cellulases are generally modular proteins with independent catalytic and cellulose-binding domain (CBD) modules and, in some bacteria, catalytic modules are noncovalently assembled on a scaffold protein with CBD to form a giant protein complex called a cellulosome, which efficiently degrades water-insoluble hard materials. In this study, a catalytic module and CBD are independently prepared by recombinant means, and are heterogeneously clustered on streptavidin and on inorganic nanoparticles for the construction of artificial cellulosomes. Heteroclustering of the catalytic module with CBD results in significant improvements in the enzyme's degradation activity for water-insoluble substrates. In particular, the increase of CBD valency in the cluster structure critically enhances the catalytic activity by improving the affinity for substrates, and clustering with multiple CBDs on CdSe nanoparticles generates a 7.2-fold increase in the production of reducing sugars relative to that of the native free enzyme. The multivalent design of substrate-binding domain on clustered cellulases is important for the construction of the artificial cellulosome, and the nanoparticles are an effective scaffold for increasing the valence of CBD in clustered cellulases. A new design is proposed for artificial cellulosomes with multiple CBDs on noncellulosome-derived scaffold structures.