Investigating the Mechanism of Action of Diketopiperazines Inhibitors of the Burkholderia cenocepacia Quorum Sensing Synthase CepI: A Site-Directed Mutagenesis Study

Abstract

Quorum sensing (QS) is a bacterial intercellular communication process which controls the production of major virulence factors, such as proteases, siderophores, and toxins, as well as biofilm formation. Since the inhibition of this pathway reduces bacterial virulence, QS is considered a valuable candidate drug target, particularly for the treatment of opportunistic infections, such as those caused by Burkholderia cenocepacia in cystic fibrosis patients. Diketopiperazine inhibitors of the acyl homoserine lactone synthase CepI have been recently described. These compounds are able to impair the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm, being also active in a Caenorhabditis elegans infection model. However, the precise mechanism of action of the compounds, as well as their effect on the cell metabolism, fundamental for candidate drug optimization, are still not completely defined. Here, we performed a proteomic analysis of B. cenocepacia cells treated with one of these inhibitors, and compared it with a cepI deleted strain. Our results demonstrate that the effects of the compound are similar to the deletion of cepI, clearly confirming that these molecules function as inhibitors of the acyl homoserine lactone synthase. Moreover, to deepen our knowledge about the binding mechanisms of the compound to CepI, we exploited previously published in silico structural insights about this enzyme structure and validated different candidate binding pockets on the enzyme surface using site-directed mutagenesis and biochemical analyses. Our experiments identified a region near the predicted S-adenosylmethionine binding site critically involved in interactions with the inhibitor. These results could be useful for future structure-based optimization of these CepI inhibitors.

Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms

“…S3). This suggests that the Cys residue at position 131 is somehow involved in the binding of BH, despite its location in a region of the enzyme far from the binding site of other CepI inhibitors (39). Using qPCR, we investigated the expression of cepI (BCAM1870) and two QS-regulated genes (aidA [BCAS0293] and zmpA [BCAS0409]) (38) in cultures derived from the TOB-exposed biofilm of lineage 2.…”

Chemical, Metabolic, and Cellular Characterization of a FtsZ Inhibitor Effective Against Burkholderia cenocepacia

“…During the last few years, we focused our attention on this topic, proposing new targets, such as the glutamate racemase (Israyilova et al, 2016), new strategies, such as the inhibition of quorum sensing (Scoffone et al, 2016;Buroni et al, 2018) and new drugs (Scoffone et al, 2014), to fight B. cenocepacia infections. We found that the benzothiadiazole derivative C109 is highly effective against B. cenocepacia (Scoffone et al, 2015) and other Gram-negative and-positive bacteria, including Mycobacterium abscessus (Hogan et al, 2018).…”

Regulatory Mechanisms and Promising Applications of Quorum Sensing-Inhibiting Agents in Control of Bacterial Biofilm Formation

“…The binding site of TofI can be occupied by an AHL analog and therefore disturb AHL synthesis ( Chung et al, 2011 ). Another example is diketopiperazines that target CepI, an AHL synthase of B. cenocepacia , can interfere with the activity of signaling molecule synthases, rendering the bacteria unable to produce biofilms ( Buroni et al, 2018 ).…”