Incorporating LsrK AI‐2 quorum quenching capability in a functionalized biopolymer capsule

Abstract

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.

Interference in Bacterial Quorum Sensing: A Biopharmaceutical Perspective

“…In addition to the studies using AHL-based QS quenchers, a recent report described the use of the AI-2 processing kinase LsrK. This enzyme was attached to a capsule of biological polymers chitosan and alginate supplemented with ATP substrate and reduced AI-2 mediated QS (Rhoads et al, 2017 ).…”

Modification and Assembly of a Versatile Lactonase for Bacterial Quorum Quenching

“…We subsequently estimated that the quantity of Sso Pox-Tyr bound to chitosan when incubated with 200 pmol was ~60 pmol. This quantity is far greater than that estimated assuming a uniformly packed monolayer of protein assembled onto the bottom of a flat well (~2 pmol [ 39 ]).…”

“…After incubation, the wells were washed and additional readings were taken (represented by the “Post-Wash” illustration). At the same time, fluorescence of samples with known quantities of labeled but unbound Sso Pox-Tyr were taken to create a calibration model and to estimate the quantity of bound Sso Pox-Tyr when incubated with ~150–200 pmol [ 39 ]. We subsequently estimated that the quantity of Sso Pox-Tyr bound to chitosan when incubated with 200 pmol was ~60 pmol.…”

“…Conversely, we estimated that a single layer of either Sso Pox-Tyr or Sso Pox-Gln would comprise ~7.6 pmol/cm 2 , or 1.9 pmol total. To make this calculation we made projected area-based estimates of a monolayer of protein (sphere) on a flat surface (chitosan) [ 39 ]. This took into consideration closest circle packing densities for a coverage of 78%, and did not consider repulsion of the similarly charged molecules.…”

“…Construction of the functionalized capsules is based on the polyelectrolyte character of chitosan and alginate bilayers enabling their self-assembly [ 43 ]. While layer-by-layer systems have been constructed, we used a one-step procedure [ 39 ]. ATP (10 mM) was mixed with alginate (2.5%) for final concentration of 222 µM ATP, vortexed, and added drop-wise by syringe needle (27 G) to a magnetically-stirred solution of 1.1% ( w / v ) chitosan and 0.27% ( w / v ) CaCl 2 .…”