IDENTIFICATION AND CHARACTERIZATION OF SMALL-MOLECULE MODULATORS OF SECONDARY METABOLISM IN BURKHOLDERIA THAILANDENSIS AND PSEUDOMONAS AERUGINOSA

Abstract

Natural products have traditionally served as a dominant source of therapeutic agents. They are produced by dedicated biosynthetic gene clusters that assemble complex, bioactive molecules from simple precursors. Recent genome sequencing efforts coupled with advances in bioinformatics indicate that the majority of biosynthetic gene clusters are not expressed under normal laboratory conditions. Termed ‘silent’ or ‘cryptic’, these gene clusters represent a treasure trove for discovery of novel small molecules, their regulatory circuits and their biosynthetic pathways. We sought to develop methods for the identification of exogenous small molecules that are able to induce expression of these clusters.

Previous efforts in our lab have outlined a strategy towards that goal, and identified trimethoprim as a global activator of secondary metabolism in Burkholderia thailandensis, a model strain for the pathogen Burkholderia pseudomallei. We have expanded upon this finding by fully characterizing the trimethoprim-induced secondary metabolome, aided by mass spectral networking. We identified a plethora of known and novel analogs for previously discovered natural products from this bacterium. In addition, we isolated and characterized a new family of natural products, the acybolins.

Our elicitor identification methodology was further applied to eight additional silent gene clusters in B. thailandensis. The resulting dataset was combined to create a heat map of ~770 elicitors and their effects on the expression of those clusters. We used this heat map to investigate what types of molecules generally act as elicitors in this strain; the major groups in this case were topoisomerase inhibitors, b-lactam antibiotics, antidepressants, antipsychotics, and antifungals. We began investigating the mode of

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action and metabolic effects of the b-lactam antibiotics and found a link to the SOS response.

Finally, we pushed our methodology still further and used it to find suppressors of virulence factor expression in Pseudomonas aeruginosa. We identified an antihypertensive drug, guanfacine, that effectively suppressed a subset of the virulence factors monitored. We posit a possible mode of action for this drug.

These efforts highlight the power of using small, bioactive molecules to probe secondary metabolism. We hope to continue using the knowledge gained in these studies to explore the nature and regulation of cryptic natural products.