Antibiotic resistance is a growing global health crisis. To prevent tremendous economic burden and human loss of life, new approaches for identifying novel antibiotics must be implemented. Target-oriented screening provides a novel approach for identifying bacterial systems that are important for pathogenesis and survival as well as next-generation antibiotics that precisely target pathogens without disrupting the normal gut microbiota. Bacterial microcompartments (MCPs) are protein-based organelles present in pathogenic bacteria. Their expression is linked to specific diseases, such as Salmonellosis, Crohn’s Disease, and urinary tract infections. Structurally, MCPs are composed of an outer shell made up of thousands of protein subunits and an enzymatic core used for the metabolism of niche carbon sources. Therefore, we hypothesized that a target-oriented screening approach to identify small molecules that disrupt MCP formation can serve as a tool for novel antibiotic discovery. Our approach uses a high throughput (HTP) method to screen for drugs that inhibit the assembly of 1,2 Propanediol utilization (Pdu) MCP shell proteins PduA and PduJ, which are essential for MCP assembly in Salmonella enterica serovar Typhimurium LT2. To accomplish this, E. coli cells over-expressing PduA and PduJ in a 384 well-block are presented a library of ~5,000 inhibitory small-molecules. Cells overexpressing PduA and PduJ are linked and elongated, which is a screenable phenotype identified via microscopy. Assembly of the linked and elongated phenotype was assessed using HTP microscopy, and loss of the screenable phenotype indicates inhibition of MCP formation by the particular small molecule being screened. In addition to identifying inhibitors that could be developed into therapeutics, this study can lead to the discovery of MCP shell features susceptible to drug inhibition.

Carolyn Mills

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