Final Report Summary - QUORUMPROBES (An Integrated Chemical Platform to Elucidate Eukaryotic Sensing of Bacterial Crosstalk) During the past 5 years I expanded and consolidated my research group and we established and validated the technology needed to achieve the goals of the QUORUMPROBES project. We synthesized several new probes that are used to identify quorum sensing receptors and we tested their efficacy in several biological systems – bacterial (E. coli and P. aeruginosa), fungal (C. albicans) and mammalian (mouse macrophages and bronchial and epithelial cell lines). We also developed new tools to visualize quorum sensing receptors, using a novel type of click reaction. This is a powerful and unconventional new technology that enables us to visualize proteins in general (and receptors in particular) in living cells through fluorescence microscopy, and it can be used as well to label, isolate and identify unknown receptors. In addition, while examining the stability of several of our probes, we made a serendipitous discovery and identified natural products in broccoli – isothiocyanates - as potent quorum sensing inhibitors and a prime new example of interkingdom interference with communication. These findings and insights have led to several new publications (in Bioorg. Med. Chem., J. Am. Chem. Soc., Chem. Rev. and Med. Chem. Comm.), and several more submitted and in preparation. We currently have three new diazirine based synthetic probes, with two new ones similar to the one we published (Dubinsky et al, ChemComm 2009), but the photocrosslink moiety (diazirine) at different positions. We have summarized our results including validation of the new probes in a new manuscript, published in Chem. Comm., 2013. We overcame the main problem with the identification of proteins using SILAC technology (to reduce the high background signals after photocrosslinking and click reaction, followed by affinity purification, dues to nonspecific binding to streptavidin based resin). We performed lots of optimization steps - and have successfully used the new probes, in collaboration with the group of Benjamin Cravatt at The Scripps Researh Institute, to identify the C12 binding proteins in mammalian cells and mouse cell lines. We were successful in identifying and validating a highly relevant protein which was previously shown to be involved in the immune response in human lungs. We are now in the final stages of the validation process (using knockout cells, pure protein and co-labeling experiments with specific antibodies) and we hope to send a manuscript for publication within two months. In addition we used our technology to find receptors for the karrikin plant natural product family, which we showed this year to be potent quorum sensing inhibitors in P. aeruginosa (Mandabi et al., Chem. Commun. 2014, 50, 5322-5355) and in V. cholerae. We are also examining the activities of these compounds in synergy with other quorum sensing inhibitors.In addition we finished a study on the effects of QS molecules on the yeast S. cerevisiae, and we found a highly specific stress response to the presence of 3-oxo-C12-HSL. We finished writing a manuscript detailing these findings, and we will send this out soon.
