Altering the Communication Networks of Multispecies Microbial Systems Using a Diverse Toolbox of AI-2 Analogues

There have been intensive efforts to find small molecule antagonists for bacterial quorum sensing (QS) mediated by the “universal” QS autoinducer, AI-2. Previous work has shown that linear and branched acyl analogues of AI-2 can selectively modulate AI-2 signaling in bacteria. Additionally, LsrK-dependent phosphorylated analogues have been implicated as the active inhibitory form against AI-2 signaling. We used these observations to synthesize an expanded and diverse array of AI-2 analogues, which included aromatic as well as cyclic C-1-alkyl analogues. Species-specific analogues that disrupted AI-2 signaling in <i>Escherichia coli</i> and <i>Salmonella typhimurium</i> were identified. Similarly, analogues that disrupted QS behaviors in <i>Pseudomonas aeruginosa</i> were found. Moreover, we observed a strong correlation between LsrK-dependent phosphorylation of these acyl analogues and their ability to suppress QS. Significantly, we demonstrate that these analogues can selectively antagonize QS in single bacterial strains in a physiologically relevant polymicrobial culture

Differential scanning calorimetry stability comparison.

<p>Differential scanning calorimetry stability comparison.</p

Enzymatic activity comparison (kcat and K_M).

<p>Enzymatic activity comparison (kcat and K_M).</p

Mass Spec and SEC-LS Analysis of candidate uricases.

<p>Mass Spec and SEC-LS Analysis of candidate uricases.</p

Uricase sequence identity comparison.

<p>Uricase sequence identity comparison.</p

PEGylation strategy and analysis.

<p>(A) Shows the three dimensional solvent accessible sites within the tetrameric crystal structure of <i>Arthrobacter globiformis</i> uricase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167935#pone.0167935.ref001" target="_blank">1</a>]. Each uricase monomer subunit of the tetrameric enzyme is shown in a different color, and residues selected for substitution with Cys (T11, N33, S142) are shown in yellow. (B) SDS-PAGE analysis of di-PEGylated and non-PEGylated uricase suggests that the di-PEGylated material runs at a much higher molecular weight relative to the non-PEGylated material. (C) A reverse-phase chromatography analysis of the di-PEGylated material suggests that 92.6% of the material is di-PEGylated, 4.4% mono-PEGylated, 0% unreacted and 2.7% over-PEGylated. (D) Demonstrates that the activity of di-PEGylated uricase is comparable to non-PEGylated uricase.</p

Enzymatic activity comparison.

<p>Substrate (UA) depletion assays were performed to assess uricase activity. The rate (specific activity) of UA oxidation was calculated based on a linear decrease in absorbance. Solid lines represent Michaelis-Menten kinetic fits performed in Prism GraphPad. Relative to Krystexxa^®, <i>Arthrobacter globiformis</i> uricase had a 2 fold better kcat and <i>Deinococcus geothermalis</i> uricase had a 2 fold better K_M (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167935#pone.0167935.t004" target="_blank">Table 4</a>).</p

Enzymatic activity comparison of RGD and SGD containing uricases.

<p>Substrate (UA) depletion assays were performed to assess uricase activity. The rate (specific activity) of UA oxidation was calculated based on a linear decrease in absorbance. Solid lines represent Michaelis-Menten kinetic fits performed in Prism GraphPad. Both RGD and SGD containing <i>Arthrobacter globiformis</i> uricases appear to have comparable enzymatic activity.</p

In vivo rat PK comparison of di and tri-PEGylated uricases.

<p>In vivo rat PK comparison of di and tri-PEGylated uricases.</p