ABSTRACT
This work sets out to provide a self-assembled biopolymer capsule activated with a multi-functional enzyme for localized delivery. This enzyme, SsoPox, which is a lactonase and phosphotriesterase, provides a means of interrupting bacterial communication pathways that have been shown to mediate pathogenicity. Here we demonstrate the capability to express, purify and attach SsoPox to the natural biopolymer chitosan, preserving its activity to "neutralize" long-chain autoinducer-1 (AI-1) communication molecules. Attachment is shown via non-specific binding and by engineering tyrosine and glutamine affinity 'tags' at the C-terminus for covalent linkage. Subsequent degradation of AI-1, in this case N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL), serves to "quench" bacterial quorum sensing (QS), silencing intraspecies communication. By attaching enzymes to pH-responsive chitosan that, in turn, can be assembled into various forms, we demonstrate device-based flexibility for enzyme delivery. Specifically, we have assembled quorum-quenching capsules consisting of an alginate inner core and an enzyme "decorated" chitosan shell that are shown to preclude bacterial QS crosstalk, minimizing QS mediated behaviors.
Subject(s)
Aryldialkylphosphatase/chemistry , Aryldialkylphosphatase/metabolism , Bacteria/enzymology , Bacterial Physiological Phenomena , Quorum Sensing , Aryldialkylphosphatase/isolation & purification , Chitosan/chemistry , Chitosan/metabolism , Enzyme Activation , Enzymes, Immobilized , Models, Molecular , Protein ConformationABSTRACT
Antibacterial resistance is an issue of increasing severity as current antibiotics are losing their effectiveness and fewer antibiotics are being developed. New methods for combating bacterial virulence are required. Modulating molecular communication among bacteria can alter phenotype, including attachment to epithelia, biofilm formation, and even toxin production. Intercepting and modulating communication networks provide a means to attenuate virulence without directly interacting with the bacteria of interest. In this work, we target communication mediated by the quorum sensing (QS) bacterial autoinducer-2, AI-2. We have assembled a capsule of biological polymers alginate and chitosan, attached an AI-2 processing kinase, LsrK, and provided substrate, ATP, for enzymatic alteration of AI-2 in culture fluids. Correspondingly, AI-2 mediated QS activity is diminished. All components of this system are "biofabricated"-they are biologically derived and their assembly is accomplished using biological means. Initially, component quantities and kinetics were tested as assembled in microtiter plates. Subsequently, the identical components and assembly means were used to create the "artificial cell" capsules. The functionalized capsules, when introduced into populations of bacteria, alter the dynamics of the AI-2 bacterial communication, attenuating QS activated phenotypes. We envision the assembly of these and other capsules or similar materials, as means to alter QS activity in a biologically compatible manner and in many environments, including in humans.
