Alginate lyase exhibits catalysis-independent biofilm dispersion and antibiotic synergy.

More than 2 decades of study support the hypothesis that alginate lyases are promising therapeutic candidates for treating mucoid Pseudomonas aeruginosa infections. In particular, the enzymes' ability to degrade alginate, a key component of mucoid biofilm matrix, has been the presumed mechanism by which they disrupt biofilms and enhance antibiotic efficacy. The systematic studies reported here show that, in an in vitro model, alginate lyase dispersion of P. aeruginosa biofilms and enzyme synergy with tobramycin are completely decoupled from catalytic activity. In fact, equivalent antibiofilm effects can be achieved with bovine serum albumin or simple amino acids. These results provide new insights into potential mechanisms of alginate lyase therapeutic activity, and they should motivate a careful reexamination of the fundamental assumptions underlying interest in enzymatic biofilm dispersion.

Toward the redesign of nutrition delivery.

In the facilitation of widespread access to low-cost, good tasting food, the global food system has relied on the use of fat, sugar, chemical processing aids and plastics, among other elements potentially detrimental to human health and the environment. This contrasts starkly with the strategies of natural nutrition delivery systems. Rich in vitamins, minerals, and other substances of functional benefit to human health, natural delivery systems, such as fruits and vegetables, retain their physical and chemical stability in a range of conditions over relatively long times through protective skins and shells that can either be eaten or degrade rapidly and fully in nature. Frequently natural foods can be delivered in small (even extremely small) portions, as with berries, insects, plankton and krill, permitting portion control and the rapid and efficient delivery of functional nutrition in inherently mobile circumstances. These and other qualities, which have insured the sustainable and healthy nourishment of animals and humans for at least tens of thousands of years, are often absent from today's man-made food and beverage delivery systems. With growing awareness of the liabilities to maintaining the food system of today, efforts are now underway to redesign nutrition delivery so as to provide the contemporary benefits of global access while retrieving the health and environmental benefits associated with natural delivery systems. We review these here, with special attention to recently commercialized nutritional delivery systems emerging from the drug delivery field aimed at reducing waste in food and beverage (nutritional aerosols) and eliminating waste in food and beverage packaging (edible skins). We briefly discuss the potential ramifications to how we will eat tomorrow.

Engineering Escherichia coli for soluble expression and single step purification of active human lysozyme.

Genetically engineered variants of human lysozyme represent promising leads in the battle against drug-resistant bacterial pathogens, but early stage development and testing of novel lysozyme variants is constrained by the lack of a robust, scalable and facile expression system. While wild type human lysozyme is reportedly produced at 50­80 kg per hectare of land in recombinant rice, this plant-based system is not readily scaled down to bench top production, and it is therefore not suitable for development and characterization of novel lysozyme variants. Here, we describe a novel and efficient expression system capable of producing folded, soluble and functional human lysozyme in Escherichia coli cells. To achieve this goal, we simultaneously co-express multiple protein folding chaperones as well as harness the lysozyme inhibitory protein, Ivy. Our strategy exploits E. coli's ease of culture, short doubling time, and facile genetics to yield upwards of 30 mg/l of soluble lysozyme in a bioreactor system, a 3000-fold improvement over prior efforts in E. coli. Additionally, molecular interactions between lysozyme and a his-tagged Ivy allows for one-step purification by IMAC, yielding as much as 21 mg/l of purified enzyme. We anticipate that our expression and purification platform will facilitate further development of engineered lysozymes having utility in disease treatment and other practical applications.

Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

Enhanced antimicrobial activity of engineered human lysozyme.

Lysozymes contain a disproportionately large fraction of cationic residues, and are thereby attracted toward the negatively charged surface of bacterial targets. Importantly, this conserved biophysical property may inhibit lysozyme antibacterial function during acute and chronic infections. A mouse model of acute pulmonary Pseudomonas aeruginosa infection demonstrated that anionic biopolymers accumulate to high concentrations in the infected lung, and the presence of these species correlates with decreased endogenous lysozyme activity. To develop antibacterial enzymes designed specifically to be used as antimicrobial agents in the infected airway, the electrostatic potential of human lysozyme (hLYS) was remodeled by protein engineering. A novel, high-throughput screen was implemented to functionally interrogate combinatorial libraries of charge-engineered hLYS proteins, and variants with improved bactericidal activity were isolated and characterized in detail. These studies illustrate a general mechanism by which polyanions inhibit lysozyme function, and they are the first direct demonstration that decreasing hLYS's net cationic character improves its antibacterial activity in the presence of disease-associated biopolymers. In addition to avoiding electrostatic sequestration, at least one charge-engineered variant also kills bacteria more rapidly in the absence of inhibitory biopolymers; this observation supports a novel hypothesis that tuning the cellular affinity of peptidoglycan hydrolases may be a general strategy for improving kinetics of bacterial killing.

Human telomerase RNA pseudoknot and hairpin thermal stability with glycine betaine and urea: preferential interactions with RNA secondary and tertiary structures.

Thermal denaturation of the human telomerase RNA (hTR) DeltaU177 pseudoknot and hTR p2b hairpin was investigated by dual UV-wavelength absorbance spectroscopy in aqueous glycine betaine and urea solutions. The hTR DeltaU177 pseudoknot contains two helix-loop interactions that comprise the tertiary structure, as well as a GC-rich 6 bp stem (stem 1) and an AU-rich 9 bp stem (stem 2). The p2b hairpin also contains GC-rich stem 1 and a unique uridine-rich helix with a pentaloop. Glycine betaine stabilizes the pseudoknot tertiary structure in 135 mm NaCl and facilitates only a minor destabilization of tertiary structure in 40 mm NaCl. As with double-helical DNA, glycine betaine interacts more strongly with the surface area exposed upon unfolding of GC-rich stem 1 than either AU-rich stem 2 or the hairpin uridine-rich helix. Urea was shown to destabilize all RNA pseudoknot and hairpin secondary and tertiary structures but exhibits a stronger preferential interaction with AU-rich stem 2. Correlating these interactions with water-accessible surface area calculations indicates that the extent of interaction of glycine betaine with the surface area exposed upon RNA unfolding decreases as the nonpolar character of the unfolded RNA surface increases. As expected, the extent of interaction of urea with the surface area exposed for unfolding RNA increases as the fraction of amide functional groups increases. However, interaction of urea with amide functional groups alone cannot explain the stronger preferential interaction of urea with AU-rich stem 2. Interaction of urea with adenine relative to guanine and cytosine bases or sequence-dependent hydration is proposed for the stronger preferential interaction of urea with AU-rich duplexes.