We developed a new approach to facilitate identification of natural products with antibacterial activities that rapidly discriminates between different mechanisms of action (MOA). This approach, bacterial cytological profiling (BCP), uses quantitative fluorescence microscopy to measure the effects of antibiotic treatment on individual cells. Antibiotics that target different cellular pathways and different steps within a pathway generate unique cytological profiles, allowing identification of the likely MOA of new compounds within a few hours. We have now developed a complimentary approach that will allow us to identify molecules that inhibit proteins that are not currently targeted by antibacterial drugs. Ths approach, which we call rapid inhibition profiling (R.I.P.), entails the rapid, inducible depletionof a target protein, followed by cytological profiling. Our preliminary data demonstrate that depletion of a drug target by R.I.P. produces cytological effects identical to those produced by the corresponding drug. Furthermore, depletion of essential proteins that are not targeted by current antibacterial drugs produces novel cytological profiles that can be subsequently used to identify molecules that inhibit these new targets. We here propose to more fully develop the R.I.P. technology by employing it on a genome wide basis in E. coli and B. subtilis. This will create a comprehensive reference set of profiles associated with the inhibition of essential cellular pathways that are not the targets of current antibacterial drugs. The genome-wide R.I.P. analysis will also provide insight into the function of conserved genes and our preliminary data suggests that it will provide insight into proteins that coordinate two or more biosynthetic pathways, providing interesting starting points for future basic research. We will then use this more complete reference data set with BCP to screen a unique and diverse collection of natural product extracts to identify those that inhibit these new drug targets and kill multidrug resistant bacteria. Together with our collaborators at Fundaci?n Medina, we will then purify and characterize our highest priority lead molecules (those that kill multidrug resistant bacteria) wit a goal of advancing them into toxicity trials.

Public Health Relevance

We have developed an approach for rapidly identifying natural products with antibacterial activities in crude natural product extracts and for rapidly discriminating between different mechanisms of action, detecting detect nuisance compounds and multiple activities in partially purified or crude natural product extracts. We have now developed a complimentary approach (R.I.P.) that will allow us to identify molecules that inhibit proteins that are not currently targeted by antibacterial drugs. We here propose to fully develop the R.I.P. technology and use it screen for antibacterial molecules and identify those that inhibit new drug targets, allowing the more rapid discovery of antibiotics to combat drug resistant bacterial pathogens.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Xu, Zuoyu
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University of California San Diego
Schools of Arts and Sciences
La Jolla
United States
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Kumaraswamy, Monika; Do, Carter; Sakoulas, George et al. (2018) Listeria monocytogenes endocarditis: case report, review of the literature, and laboratory evaluation of potential novel antibiotic synergies. Int J Antimicrob Agents 51:468-478
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Kumaraswamy, Monika; Lin, Leo; Olson, Joshua et al. (2016) Standard susceptibility testing overlooks potent azithromycin activity and cationic peptide synergy against MDR Stenotrophomonas maltophilia. J Antimicrob Chemother 71:1264-9
Lin, Leo; Nonejuie, Poochit; Munguia, Jason et al. (2015) Azithromycin Synergizes with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity Against Highly Multidrug-Resistant Gram-Negative Bacterial Pathogens. EBioMedicine 2:690-8

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