Modification and Assembly of a Versatile Lactonase for Bacterial Quorum Quenching

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.

Bacterial co-culture with cell signaling translator and growth controller modules for autonomously regulated culture composition

“…To clone pAHL-HPr, sfGFP was replaced with ptsH in a previously cloned AI-1 fluorescent reporter plasmid (pAHL-Reporter_Red-Green) with constitutive expression of dsRedExpress2 42 . Primers HPr-SpeI-F and HPr-SacI-R were used to amplify ptsH while adding SpeI and SacI restriction digestion sites upstream and downstream of the start and stop codons, respectively.…”

“…Experimental CM samples were prepared by filtering supernatant through a 0.2 µM filter and storing at −20 °C until needed for activity assays. For the AI-1 activity assay 42 , E. coli luminescent reporter cells containing plasmid pAL105 45 were grown in LB media overnight. In the morning they were diluted 2500 fold in LB media with 50 µg mL − 1 tetracycline and 50 µg mL −1 kanamycin.…”

Capsules with bacteria and fungi in distinct compartments: A platform for studying microbes from different kingdoms and their cross-communication

“…We used a reporter strain of E . coli that produce bioluminescence proportional to the AI-1 present (see Experimental Section for details) [ 37 ]. From this assay, we measured the AI-1 concentration in the culture media to be 19 μM.…”

“…A bioluminescent reporter assay was used to measure the AI-1 produced by P . aeruginosa in the microcapsules [ 37 ]. The microcapsules were incubated for 18 h and the surrounding media was collected, filtered through a 0.2 μM filter, and stored at −20°C until needed.…”

“…For this, we measured how much AI-1 was present in the culture media after 12 h of culture in a sample with 50 MCCs (each with B and F compartments, as shown in Fig 8). We used a reporter strain of E. coli that produce bioluminescence proportional to the AI-1 present (see Experimental Section for details) [37]. From this assay, we measured the AI-1 concentration in the culture media to be 19 μM.…”